The Impact of Mutations in SARS-CoV-2 Spike on Viral Infectivity and Antigenicity.

The spike protein of SARS-CoV-2 has been undergoing mutations and is highly glycosylated. It is critically important to investigate the biological significance of these mutations. Here, we investigated 80 variants and 26 glycosylation site modifications for the infectivity and reactivity to a panel of neutralizing antibodies and sera from convalescent patients. D614G, along with several variants containing both D614G and another amino acid change, were significantly more infectious. Most variants with amino acid change at receptor binding domain were less infectious, but variants including A475V, L452R, V483A, and F490L became resistant to some neutralizing antibodies. Moreover, the majority of glycosylation deletions were less infectious, whereas deletion of both N331 and N343 glycosylation drastically reduced infectivity, revealing the importance of glycosylation for viral infectivity. Interestingly, N234Q was markedly resistant to neutralizing antibodies, whereas N165Q became more sensitive. These findings could be of value in the development of vaccine and therapeutic antibodies.

SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2.

We show that SARS-CoV-2 spike protein interacts with both cellular heparan sulfate and angiotensin-converting enzyme 2 (ACE2) through its receptor-binding domain (RBD). Docking studies suggest a heparin/heparan sulfate-binding site adjacent to the ACE2-binding site. Both ACE2 and heparin can bind independently to spike protein in vitro, and a ternary complex can be generated using heparin as a scaffold. Electron micrographs of spike protein suggests that heparin enhances the open conformation of the RBD that binds ACE2. On cells, spike protein binding depends on both heparan sulfate and ACE2. Unfractionated heparin, non-anticoagulant heparin, heparin lyases, and lung heparan sulfate potently block spike protein binding and/or infection by pseudotyped virus and authentic SARS-CoV-2 virus. We suggest a model in which viral attachment and infection involves heparan sulfate-dependent enhancement of binding to ACE2. Manipulation of heparan sulfate or inhibition of viral adhesion by exogenous heparin presents new therapeutic opportunities.

V-CARMA: A tool for the detection and modification of antigen-specific T cells.

T cells promote our body's ability to battle cancers and infectious diseases but can act pathologically in autoimmunity. The recognition of peptides presented by major histocompatibility complex (pMHC) molecules by T cell receptors (TCRs) enables T cell-mediated responses. To modify disease-relevant T cells, new tools to genetically modify T cells and decode their antigen recognition are needed. Here, we present an approach using viruses pseudotyped with peptides loaded on MHC called V-CARMA (Viral ChimAeric Receptor MHC-Antigen) to specifically target T cells expressing cognate TCRs for antigen discovery and T cell engineering. We show that lentiviruses displaying antigens on human leukocyte antigen (HLA) class I and class II molecules can robustly infect CD8+ and CD4+ T cells expressing cognate TCRs, respectively. The infection rates of the pseudotyped lentiviruses (PLVs) are correlated with the binding affinity of the TCR to its cognate antigen. Furthermore, peptide-HLA pseudotyped lentivirus V-CARMA constructs can identify target cells from a mixed T cell population, suppress PD-1 expression on CD8+ T cells via PDCD1 shRNA delivery, and induce apoptosis in autoreactive CD4+ T cells. Thus, V-CARMA is a versatile tool for TCR ligand identification and selective T cell manipulation.

Evaluating long-term antibody responses to booster doses of COVID-19 vaccines in the Pakistani population.

Background & Objective: Nearly 80 million of the Pakistani population received two doses of the BBIBP-CorV vaccine, against SARS-CoV-2, and 2.6 million people received heterologous booster doses up to February 2022. Our objective was to measure the long-term change of antibody titers in persons vaccinated with Pfizer-BioNTech COVID-19 following two doses of BBIBP-CorV. Methods: Serum specimens from forty-three participants were collected 4-8 weeks following two doses of BBIBP-CorV at the Indus Hospital & Health Network, Karachi. A second set of serum specimens were collected 2-12 months after Pfizer-BioNTech COVID-19 booster dose administration. Chemiluminescent Microparticle Immunoassay (CMIA, Abbott Alinity Quant), and the pseudotyped lentivirus antibody neutralization assay were performed on all specimens. The latter assay was reported as log half-maximal inhibitory concentrations (IC50), calculated using a nonlinear regression algorithm (log [inhibitor] versus normalized response variable slope) in Graph Pad Prism 9. Paired sample t-test was used to ascertain the statistical significance of the difference in means of antibody titers obtained before and after the booster vaccine doses. Results: Mean log10 values obtained with CMIA before and after the booster dose were 2.90 AU/mL and 3.87 AU/mL respectively, while the corresponding log10 IC50 values obtained through pseudotyped lentivirus antibody neutralization assay were 2.45 and 2.80. These differences were statistically significant with CMIA (p = <0.00001), but not with pseudotyped lentivirus antibody neutralization assay (p = 0.06318.). Conclusion: A heterologous booster dose with Pfizer-BioNTech COVID-19 vaccine following two doses of BBIBP results in increased total antibody titers, though neutralizing antibody titers may start to wane a few months after the booster dose.

Selective, broad-spectrum antiviral photodynamic disinfection with dicationic imidazolyl chlorin photosensitizers.

The COVID-19 pandemic exposes our vulnerability to viruses that acquire the ability to infect our cells. Classical disinfection methods are limited by toxicity. Existing medicines performed poorly against SARS-CoV-2 because of their specificity to targets in different organisms. We address the challenge of mitigating known and prospective viral infections with a new photosensitizer for antimicrobial photodynamic therapy (aPDT). Photodynamic inactivation is based on local oxidative stress, which is particularly damaging to enveloped viruses. We synthesized a cationic imidazolyl chlorin that reduced by > 99.999% of the percentage inhibition of amplification of SARS-CoV-2 collected from patients at 0.2 µM concentration and 4 J cm-2. Similar results were obtained in the prevention of infection of human ACE2-expressing HEK293T cells by a pseudotyped lentiviral vector exhibiting the S protein of SARS-CoV-2 at its surface. No toxicity to human epidermal keratinocytes (HaCaT) cells was found under similar conditions. aPDT with this chlorin offers fast and safe broad-spectrum photodisinfection and can be repeated with low risk of resistance.

Pseudotyped Viruses for Orthohantavirus.

Orthohantaviruses, members of the Orthohantavirus genus of Hantaviridae family of the Bunyavirales order, are enveloped, negative-sense, single-stranded, tripartite RNA viruses. They are emerging zoonotic pathogens carried by small mammals including rodents, moles, shrews, and bats and are the etiologic agents of hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) among humans. With the characteristics of low biological risk but strong operability, a variety of pseudotyped viruses have been constructed as alternatives to authentic orthohantaviruses to help delineate the roles of host factors in viral entry and other virus-host interactions, to assist in deciphering mechanisms of immune response and correlates of protection, to enhance our understanding of viral antigenic property, to characterize viral entry inhibitors, and to be developed as vaccines. In this chapter, we will discuss the general property of orthohantavirus, construction of pseudotyped orthohantaviruses based on different packaging systems, and their current applications.

Pseudotyped Viruses for Enterovirus.

Using a non-pathogenic pseudotyped virus as a surrogate for a wide-type virus in scientific research complies with the recent requirements for biosafety. Enterovirus (EV) contains many species of viruses, which are a type of nonenveloped virus. The preparation of its corresponding pseudotyped virus often needs customized construction compared to some enveloped viruses. This article describes the procedures and challenges in the construction of pseudotyped virus for enterovirus (pseudotyped enterovirus, EVpv) and also introduces the application of EVpv in basic virological research, serological monitoring, and the detection of neutralizing antibody (NtAb).

Pseudotyped Viruses for the Alphavirus Chikungunya Virus.

Members of the genus Alphavirus are mostly mosquito-borne pathogens that cause disease in their vertebrate hosts. Chikungunya virus (CHIKV), which is one member of the genus Alphavirus [1], has been a major health problem in endemic areas since its re-emergence in 2006. CHIKV is transmitted to mammalian hosts by the Aedes mosquito, causing persistent debilitating symptoms in many cases. At present, there is no specific treatment or vaccine. Experiments involving live CHIKV need to be performed in BSL-3 facilities, which limits vaccine and drug research. The emergence of pseudotyped virus technology offered the potential for the development of a safe and effective evaluation method. In this chapter, we review the construction and application of pseudotyped CHIKVs, the findings from which have enhanced our understanding of CHIKV. This will, in turn, enable the exploration of promising therapeutic strategies in animal models, with the ultimate aim of developing effective treatments and vaccines against CHIKV and other related viruses.

Pseudotyped Virus for Papillomavirus.

Papillomavirus is difficult to culture in vitro, which limits its related research. The development of pseudotyped virus technology provides a valuable research tool for virus infectivity research, vaccine evaluation, infection inhibitor evaluation, and so on. Depending on the application fields, different measures have been developed to generate various kinds of pseudotyped papillomavirus. L1-based and L2-based HPV vaccines should be evaluated using different pseudotyped virus system. Pseudotyped papillomavirus animal models need high-titer pseudotyped virus and unique handling procedure to generate robust results. This paper reviewed the development, optimization, standardization, and application of various pseudotyped papillomavirus methods.

Pseudotyped Virus for Henipavirus.

The genus Henipavirus (HNV) includes two virulent infectious viruses, Nipah virus (NiV) and Hendra virus (HeV), which are the focus of considerable public health research efforts and have been classified as priority infectious diseases by the World Health Organization. Both viruses are high risk and should be handled in biosafety level 4 laboratories. Pseudotyped viruses containing the envelope proteins of HNV viruses have the same envelope protein structure as the authentic viruses; thus, they can mimic the receptor-binding and membrane fusion processes of authentic viruses with host cells and can be handled in biosafety level 2 laboratories. These characteristics enable pseudotyped viruses to be widely used in studies of viral infection mechanisms (packaging, budding, virus attachment, membrane fusion, viral entry, and glycosylation), inhibitory drug screening assays, and monoclonal antibody neutralization characteristics. This review will provide an overview of the progress of research concerning pseudotyped virus packaging systems for NiV and HeV.

Assays Based on Pseudotyped Viruses.

Pseudotyped viruses are more and more widely used in virus research and the evaluation of antiviral products because of their high safety, simple operation, high accessibility, ease in achieving standardization, and high throughput. The development of measures based on pseudotyped virus is closely related to the characteristics of viruses, and it is also necessary to follow the principles of assay development. Only in the process of method development, where the key parameters that affect the results are systematically optimized and the preliminary established method is fully validated, can the accuracy, reliability, and repeatability of the test results be ensured. Only the method established on this basis can be transferred to different laboratories and make the results of different laboratories comparable. This paper summarizes the specific aspects and general principles in the development of assays based on pseudotyped virus, which is of reference value for the development of similar methods.

Pseudotyped Viruses.

Pseudotyped viruses have been constructed for many viruses. They can mimic the authentic virus and have many advantages compared to authentic viruses. Thus, they have been widely used as a surrogate of authentic virus for viral function analysis, detection of neutralizing antibodies, screening viral entry inhibitors, and others. This chapter reviewed the progress in the field of pseudotyped viruses in general, including the definition and the advantages of pseudotyped viruses, their potential usage, different strategies or vectors used for the construction of pseudotyped viruses, and factors that affect the construction of pseudotyped viruses.

Application of Pseudotyped Viruses.

Highly pathogenic emerging and reemerging viruses have serious public health and socioeconomic implications. Although conventional live virus research methods can more reliably investigate disease pathogenicity and evaluate antiviral products, they usually depend on high-level biosafety laboratories and skilled researchers; these requirements hinder in vitro assessments of efficacy, as well as efforts to test vaccines and antibody drugs. In contrast, pseudotyped viruses (i.e., single-round infectious viruses that mimic the membrane structures of various live viruses) are widely used in studies of highly pathogenic viruses because they can be handled in biosafety level 2 facilities. This chapter provides a concise overview of various aspects of pseudotyped virus technologies, including (1) exploration of the mechanisms of viral infection; (2) evaluation of the efficacies of vaccines and monoclonal antibodies based on pseudovirion-based neutralization assay; (3) assessment of antiviral agents (i.e., antibody-based drugs and inhibitors); (4) establishment of animal models of pseudotyped virus infection in vivo; (5) investigation of the evolution, infectivity, and antigenicity of viral variants and viral glycosylation; and (6) prediction of antibody-dependent cell-mediated cytotoxic activity.

Pseudotyped Viruses for Retroviruses.

Since the discovery of retroviruses, their genome and replication strategies have been extensively studied, leading to the discovery of several unique features that make them invaluable vectors for virus pseudotyping, gene delivery, and gene therapy. Notably, retroviral vectors enable the integration of a gene of interest into the host genome, they can be used to stably transduce both dividing and nondividing cells, and they can deliver relatively large genes. Today, retroviral vectors are commonly used for many research applications and have become an active tool in gene therapy and clinical trials. This chapter will discuss the important features of the retroviral genome and replication cycle that are crucial for the development of retroviral vectors, the different retrovirus-based vector systems that are commonly used, and finally the research and clinical applications of retroviral vectors.

Pseudotyped Viruses for Phlebovirus.

Rift Valley fever virus (RVFV) is a member of the Phlebovirus genus, one of the 20 genera in the Phenuiviridae family. RVFV causes disease in animals and humans and is transmitted by sandflies or ticks. However, research into RVFV is limited by the requirement for biosafety level 3 (BSL-3) containment. Pseudotyped virus overcomes this limitation as it can be handled in a BSL-2 environment. Pseudotyped RVFV possesses an identical envelope protein structure to that of the authentic virus, simulating the same process of receptor binding and membrane fusion to host cells. Pseudotyped phleboviruses are therefore useful tools to study the infection mechanism of these viruses and for the screening of inhibitory drugs and the development of therapeutic monoclonal antibodies.

Pseudotyped Virus for Bandavirus.

The genus Bandavirus, belonging to family Phenuiviridae, order Bunyavirales, consists of eight tick-borne bunyaviruses. The Dabie bandavirus, formerly known as severe fever with thrombocytopenia virus (SFTSV), belongs to the genus Bandavirus. This emerging pathogen was first identified in central China in 2009. In recent years, the disease has been reported to cause several outbreaks in eastern Asia areas, including China, Japan, Korea, and Vietnam. Tick-to-human transmission is the main route of infection in humans, and transmission via the contact of body fluids from person-to-person was also reported. Despite its high fatality rate, there is currently no vaccine or antiviral therapy available. The therapeutic efficacies of several antiviral agents against Dabie bandavirus are still being evaluated. However, the virus is a potent pathogen with high biosafety experimental conditions. Therefore, replication-incompetent pseudotyped viruses play an important role. In this chapter, we succinctly summarize the basic features concerning Dabie bandavirus, including virion structure, genome characteristics, especially the characteristics of glycoprotein, and probable pathogenic mechanism. And, we put an important part in expounding the construction of pseudoviruses and its application.

Pseudotyped Viruses for Coronaviruses.

Seven coronaviruses have been identified that can infect humans, four of which usually cause mild symptoms, including HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1, three of which are lethal coronaviruses, named severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus, and severe acute respiratory syndrome coronavirus 2. Pseudotyped virus is an important tool in the field of human coronavirus research because it is safe, easy to prepare, easy to detect, and highly modifiable. In addition to the application of pseudotyped viruses in the study of virus infection mechanism, vaccine, and candidate antiviral drug or antibody evaluation and screening, pseudotyped viruses can also be used as an important platform for further application in the prediction of immunogenicity and antigenicity after virus mutation, cross-species transmission prediction, screening, and preparation of vaccine strains with better broad spectrum and antigenicity. Meanwhile, as clinical trials of various types of vaccines and post-clinical studies are also being carried out one after another, the establishment of a high-throughput and fully automated detection platform based on SARS-CoV-2 pseudotyped virus to further reduce the cost of detection and manual intervention and improve the efficiency of large-scale detection is also a demand for the development of SARS-CoV-2 pseudotyped virus.

Pseudotyped Viruses for Mammarenavirus.

Mammarenaviruses are classified into New World arenaviruses (NW) and Old World arenaviruses (OW). The OW arenaviruses include the first discovered mammarenavirus-lymphocytic choriomeningitis virus (LCMV) and the highly lethal Lassa virus (LASV). Mammarenaviruses are transmitted to human by rodents, resulting in severe acute infections and hemorrhagic fever. Pseudotyped viruses have been widely used as a tool in the study of mammarenaviruses. HIV-1, SIV, FIV-based lentiviral vectors, VSV-based vectors, MLV-based vectors, and reverse genetic approaches have been applied in the construction of pseudotyped mammarenaviruses. Pseudotyped mammarenaviruses are commonly used in receptor research, neutralizing antibody detection, inhibitor screening, viral virulence studies, functional analysis of N-linked glycans, and studies of viral infection, endocytosis, and fusion mechanisms.

Isolation of Cells from Glioblastoma Multiforme Grade 4 Tumors for Infection with Zika Virus prME and ME Pseudotyped HIV-1.

This study aimed to isolate cells from grade 4 glioblastoma multiforme tumors for infection experiments with Zika virus (ZIKV) prME or ME enveloped HIV-1 pseudotypes. The cells obtained from tumor tissue were successfully cultured in human cerebrospinal fluid (hCSF) or a mixture of hCSF/DMEM in cell culture flasks with polar and hydrophilic surfaces. The isolated tumor cells as well as the U87, U138, and U343 cells tested positive for ZIKV receptors Axl and Integrin αvß5. Pseudotype entry was detected by the expression of firefly luciferase or green fluorescent protein (gfp). In prME and ME pseudotype infections, luciferase expression in U-cell lines was 2.5 to 3.5 logarithms above the background, but still two logarithms lower than in the VSV-G pseudotype control. Infection of single cells was successfully detected in U-cell lines and isolated tumor cells by gfp detection. Even though prME and ME pseudotypes had low infection rates, pseudotypes with ZIKV envelopes are promising candidates for the treatment of glioblastoma.

Bovine lactoferrin increases the poly(I:C)-induced antiviral response in vitro.

Bovine lactoferrin (bLF) is a naturally occurring glycoprotein with antibacterial and antiviral activities. We evaluated whether bLF can prevent viral infections in the human intestinal epithelial cell line Caco-2. To assess antiviral responses, we measured the levels of interferon (IFN) expression, IFN-stimulated gene expression, and infection with a pseudotyped virus bearing either severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein or vesicular stomatitis virus (VSV)-G protein after treatment of cells with both bLF and polyinosinic-polycytidylic acid, an analog of double-stranded RNA that mimics viral infection. Combination treatment of cells with both bLF and polyinosinic-polycytidylic acid increased mRNA and protein expression of several IFN genes (IFNB, IFNL1, and IFNL2) and IFN-stimulated genes (ISG15, MX1, IFITM1, and IFITM3) in Caco-2 cells. However, treatment with bLF alone did not induce an antiviral response. Furthermore, combination treatment suppressed infection of the SARS-CoV-2 pseudotyped virus more efficiently than did bLF treatment alone, even though combination treatment increased the expression of mRNA encoding ACE2. These results indicate that bLF increases the antiviral response associated with the double-stranded RNA-stimulated signaling pathway. Our results also suggest that bLF and double-stranded RNA analogs can be used to treat viral infections, including those caused by SARS-CoV-2.