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Antiviral neutralizing antibodies (nAbs) are commonly derived from B cells developed in immunized or infected animals and humans. Fully human antibodies are preferred for clinical use as they are potentially less immunogenic. However, the function of B cells varies depending on their homing pattern and an additional hurdle for antibody discovery in humans is the source of human tissues with an immunological microenvironment. Here, we show an efficient method to pharm human antibodies using immortalized B cells recovered from Nod.Rag.Gamma (NRG) mice reconstituting the human immune system (HIS). Humanized HIS mice were immunized either with autologous engineered dendritic cells expressing the human cytomegalovirus gB envelope protein (HCMV-gB) or with Epstein–Barr virus-like particles (EB-VLP). Human B cells recovered from spleen of HIS mice were efficiently immortalized with EBV in vitro. We show that these immortalized B cells secreted human IgGs with neutralization capacities against prototypic HCMV-gB and EBV-gp350. Taken together, we show that HIS mice can be successfully used for the generation and pharming fully human IgGs. This technology can be further explored to generate antibodies against emerging infections for diagnostic or therapeutic purposes.

The successful development of effective viral vaccines depends on well-known correlates of protection, high immunogenicity, acceptable safety criteria, low reactogenicity, and well-designed immune monitoring and serology. Virus-neutralizing antibodies are often a good correlate of protective immunity, and their serum concentration is a key parameter during the pre-clinical and clinical testing of vaccine candidates. Viruses are inherently infectious and potentially harmful, but we and others developed replication-defective SARS-CoV-2 virus-like-particles (VLPs) as surrogates for infection to quantitate neutralizing antibodies with appropriate target cells using a split enzyme-based approach. Here, we show that SARS-CoV-2 and Epstein–Barr virus (EBV)-derived VLPs associate and fuse with extracellular vesicles in a highly specific manner, mediated by the respective viral fusion proteins and their corresponding host receptors. We highlight the capacity of virus-neutralizing antibodies to interfere with this interaction and demonstrate a potent application using this technology. To overcome the common limitations of most virus neutralization tests, we developed a quick in vitro diagnostic assay based on the fusion of SARS-CoV-2 VLPs with susceptible vesicles to quantitate neutralizing antibodies without the need for infectious viruses or living cells. We validated this method by testing a set of COVID-19 patient serum samples, correlated the results with those of a conventional test, and found good sensitivity and specificity. Furthermore, we demonstrate that this serological assay can be adapted to a human herpesvirus, EBV, and possibly other enveloped viruses.

Understanding immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial to contain the COVID-19 pandemic. Using a multiplex approach, serum IgG responses against the whole SARS-CoV-2 proteome and the nucleocapsid proteins of endemic human coronaviruses (HCoVs) were measured in SARS-CoV-2-infected donors and healthy controls. COVID-19 severity strongly correlated with IgG responses against the nucleocapsid (N) of SARS-CoV-2 and possibly with the number of viral antigens targeted. Furthermore, a strong correlation between COVID-19 severity and serum responses against N of endemic alpha- but not betacoronaviruses was detected. This correlation was neither caused by cross-reactivity of antibodies, nor by a general boosting effect of SARS-CoV-2 infection on pre-existing humoral immunity. These findings raise the prospect of a potential disease progression marker for COVID-19 severity that allows for early stratification of infected individuals.

Epstein-Barr virus (EBV) is a double-stranded DNA virus of the Herpesviridae family. This virus preferentially infects human primary B cells and persists in the human B cell compartment for a lifetime. Latent EBV infection can lead to the development of different types of lymphomas as well as carcinomas such as nasopharyngeal and gastric carcinoma in immunocompetent and immunocompromised patients. The early phase of viral infection is crucial for EBV to establish latency, but different viral components are sensed by cellular sensors called pattern recognition receptors (PRRs) as the first line of host defense. The efficacy of innate immunity, in particular the interferon-mediated response, is critical to control viral infection initially and to trigger a broad spectrum of specific adaptive immune responses against EBV later. Despite these restrictions, the virus has developed various strategies to evade the immune reaction of its host and to establish its lifelong latency. In its different phases of infection, EBV expresses up to 44 different viral miRNAs. Some act as viral immunoevasins because they have been shown to counteract innate as well as adaptive immune responses. Similarly, certain virally encoded proteins also control antiviral immunity. In this review, we discuss how the virus governs innate immune responses of its host and exploits them to its advantage.

Epstein–Barr virus (EBV) is etiologically associated with a number of malignant and non-malignant conditions. Thus, a prophylactic vaccine against this virus could help to reduce the burden of many EBV-associated diseases. Previously, we reported that an EBV virus-like particle (VLP) vaccine was highly immunogenic and produced a strong humoral response in mice. However, since EBV does not infect mice, the efficacy of the VLP in preventing EBV infection could not be addressed. Here we examined, for the first time, the efficacy of the EBV-VLP vaccine using a novel rabbit model of EBV infection. Animals vaccinated with two doses of VLP elicited higher antibody responses to total EBV antigens compared to animals receiving one dose. Vaccinated animals also elicited both IgM and IgG to EBV-specific antigens, VCA and EBNA1. Analysis of peripheral blood and spleen for EBV copy number indicated that the viral load in both of these compartments was lower in animals receiving a 2-dose vaccine. However, the VLP vaccine was ineffective in preventing EBV infection. With several other EBV vaccine candidates currently at various stages of development and testing, we believe that the rabbit model of EBV infection could be a great platform for evaluating potential candidates.

ABSTRACT The innate immune system serves as frontline defense against pathogens, such as bacteria and viruses. Natural killer (NK) cells are a part of innate immunity and can both secrete cytokines and directly target cells for lysis. NK cells express several cell surface receptors, including NKG2D, which bind multiple ligands. People with deficiencies in NK cells are often susceptible to uncontrolled infection by herpesviruses, such as Epstein-Barr virus (EBV). Infection with EBV stimulates both innate and adaptive immunity, yet the virus establishes lifelong latent infection in memory B cells. We show that the EBV oncogene EBNA1, previously known to be necessary for maintaining EBV genomes in latently infected cells, also plays an important role in suppressing NK cell responses and cell death in newly infected cells. EBNA1 does so by downregulating the NKG2D ligands ULBP1 and ULBP5 and modulating expression of c-Myc. B cells infected with a derivative of EBV that lacks EBNA1 are more susceptible to NK cell-mediated killing and show increased levels of apoptosis. Thus, EBNA1 performs a previously unappreciated role in reducing immune response and programmed cell death after EBV infection, helping infected cells avoid immune surveillance and apoptosis and thus persist for the lifetime of the host. IMPORTANCE Epstein-Barr virus (EBV) is a ubiquitous human pathogen, infecting up to 95% of the world’s adult population. Initial infection with EBV can cause infectious mononucleosis. EBV is also linked to several human malignancies, including lymphomas and carcinomas. Although infection by EBV alerts the immune system and causes an immune response, the virus persists for life in memory B cells. We show that the EBV protein EBNA1 can downregulate several components of the innate immune system linked to natural killer (NK) cells. This downregulation of NK cell activity translates to lower killing of EBV-infected cells and is likely one way that EBV escapes immune surveillance after infection. Additionally, we show that EBNA1 reduces apoptosis in newly infected B cells, allowing more of these cells to survive. Taken together, our findings uncover new functions of EBNA1 and provide insights into viral strategies to survive the initial immune response postinfection.

Mammalian cells release different types of vesicles, collectively termed extracellular vesicles (EVs). EVs contain cellular microRNAs (miRNAs) with an apparent potential to deliver their miRNA cargo to recipient cells to affect the stability of individual mRNAs and the cells' transcriptome. The extent to which miRNAs are exported via the EV route and whether they contribute to cell-cell communication are controversial. To address these issues, we defined multiple properties of EVs and analyzed their capacity to deliver packaged miRNAs into target cells to exert biological functions. We applied well-defined approaches to produce and characterize purified EVs with or without specific viral miRNAs. We found that only a small fraction of EVs carried miRNAs. EVs readily bound to different target cell types, but EVs did not fuse detectably with cellular membranes to deliver their cargo. We engineered EVs to be fusogenic and document their capacity to deliver functional messenger RNAs. Engineered fusogenic EVs, however, did not detectably alter the functionality of cells exposed to miRNA-carrying EVs. These results suggest that EV-borne miRNAs do not act as effectors of cell-to-cell communication.