Monoclonal Antibodies to S and N SARS-CoV-2 Proteins as Probes to Assess Structural and Antigenic Properties of Coronaviruses.

Antibodies targeting the spike (S) and nucleocapsid (N) proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are essential tools. In addition to important roles in the treatment and diagnosis of infection, the availability of high-quality specific antibodies for the S and N proteins is essential to facilitate basic research of virus replication and in the characterization of mutations responsible for variants of concern. We have developed panels of mouse and rabbit monoclonal antibodies (mAbs) to the SARS-CoV-2 spike receptor-binding domain (S-RBD) and N protein for functional and antigenic analyses. The mAbs to the S-RBD were tested for neutralization of native SARS-CoV-2, with several exhibiting neutralizing activity. The panels of mAbs to the N protein were assessed for cross-reactivity with the SARS-CoV and Middle East respiratory syndrome (MERS)-CoV N proteins and could be subdivided into sets that showed unique specificity for SARS-CoV-2 N protein, cross-reactivity between SARS-CoV-2 and SARS-CoV N proteins only, or cross-reactivity to all three coronavirus N proteins tested. Partial mapping of N-reactive mAbs were conducted using truncated fragments of the SARS-CoV-2 N protein and revealed near complete coverage of the N protein. Collectively, these sets of mouse and rabbit monoclonal antibodies can be used to examine structure/function studies for N proteins and to define the surface location of virus neutralizing epitopes on the RBD of the S protein.

Th1-like Plasmodium-Specific Memory CD4+ T Cells Support Humoral Immunity.

Effector T cells exhibiting features of either T helper 1 (Th1) or T follicular helper (Tfh) populations are essential to control experimental Plasmodium infection and are believed to be critical for resistance to clinical malaria. To determine whether Plasmodium-specific Th1- and Tfh-like effector cells generate memory populations that contribute to protection, we developed transgenic parasites that enable high-resolution study of anti-malarial memory CD4 T cells in experimental models. We found that populations of both Th1- and Tfh-like Plasmodium-specific memory CD4 T cells persist. Unexpectedly, Th1-like memory cells exhibit phenotypic and functional features of Tfh cells during recall and provide potent B cell help and protection following transfer, characteristics that are enhanced following ligation of the T cell co-stimulatory receptor OX40. Our findings delineate critical functional attributes of Plasmodium-specific memory CD4 T cells and identify a host-specific factor that can be targeted to improve resolution of acute malaria and provide durable, long-term protection against Plasmodium parasite re-exposure.

Plasmodium vivax liver stage development and hypnozoite persistence in human liver-chimeric mice.

Plasmodium vivax malaria is characterized by periodic relapses of symptomatic blood stage parasite infections likely initiated by activation of dormant liver stage parasites-hypnozoites. The lack of tractable P. vivax animal models constitutes an obstacle in examining P. vivax liver stage infection and drug efficacy. To overcome this obstacle, we have used human liver-chimeric (huHep) FRG KO mice as a model for P. vivax infection. FRG KO huHep mice support P. vivax sporozoite infection, liver stage development, and hypnozoite formation. We show complete P. vivax liver stage development, including maturation into infectious exo-erythrocytic merozoites as well as the formation and persistence of hypnozoites. Prophylaxis or treatment with the antimalarial primaquine can prevent and eliminate liver stage infection, respectively. Thus, P. vivax-infected FRG KO huHep mice are a model to investigate liver stage development and dormancy and may facilitate the discovery of drugs targeting relapsing malaria.

Plasmodium yoelii inhibitor of cysteine proteases is exported to exomembrane structures and interacts with yoelipain-2 during asexual blood-stage development.

Plasmodium falciparum (Pf) blood stages express falstatin, an inhibitor of cysteine proteases (ICP), which is implicated in regulating proteolysis during red blood cell infection. Recent data using the Plasmodium berghei rodent malaria model suggested an additional role for ICP in the infection of hepatocytes by sporozoites and during liver-stage development. Here we further characterize the role of ICP in vivo during infection with Plasmodium yoelii (Py) and Pf. We found that Py-ICP was refractory to targeted gene deletion indicating an essential function during asexual blood-stage replication, but significant downregulation of ICP using a regulated system did not impact blood-stage growth. Py-ICP localized to vesicles within the asexual blood-stage parasite cytoplasm, as well as the parasitophorous vacuole, and was exported to dynamic exomembrane structures in the infected RBC. In sporozoites, expression was observed in rhoptries, in addition to intracellular vesicles distinct from TRAP containing micronemes. During liver-stage development, Py-ICP was confined to the parasite compartment until the final phase of liver-stage development when, after parasitophorous vacuolemembrane breakdown, it was released into the infected hepatocyte. Finally, we identified the cysteine protease yoelipain-2 as a binding partner of Py-ICP during blood-stage infection. These data show that ICP may be important in regulating proteolytic processes during blood-stage development, and is likely playing a role in liver stage-hepatocyte interactions at the time of exoerythrocytic merozoite release.

The affinity of EBNA1 for its origin of DNA synthesis is a determinant of the origin's replicative efficiency.

Epstein-Barr virus (EBV) replicates its genome as a licensed plasmid in latently infected cells. Although replication of this plasmid is essential for EBV latent infection, its synthesis still fails for 16% of the templates in S phase. In order to understand these failures, we sought to determine whether the affinity of the initiator protein (EBNA1) for its binding sites in the origin affects the efficiency of plasmid replication. We have answered this question by using several engineered origins modeled upon the arrangement of EBNA1-binding sites found in DS, the major plasmid origin of EBV. The human TRF2 protein also binds to half-sites in DS and increases EBNA1's affinity for its own sites; we therefore also tested origin efficiency in the presence or absence of these sites. We have found that if TRF2-half-binding sites are present, the efficiency of supporting the initiation of DNA synthesis and of establishing a plasmid bearing that origin directly correlates with the affinity of EBNA1 for that origin. Moreover, the presence of TRF2-half-binding sites also increases the average level of EBNA1 and ORC2 bound to those origins in vivo, as measured by chromatin immunoprecipitation. Lastly, we have created an origin of DNA synthesis from high-affinity EBNA1-binding sites and TRF2-half-binding sites that functions severalfold more efficiently than does DS. This finding indicates that EBV has selected a submaximally efficient origin of DNA synthesis for the latent phase of its life cycle. This enhanced origin could be used practically in human gene vectors to improve their efficiency in therapy and basic research.

The plasmid replicon of Epstein-Barr virus: mechanistic insights into efficient, licensed, extrachromosomal replication in human cells.

The genome of Epstein-Barr Virus (EBV) and plasmid derivatives of it are among the most efficient extrachromosomal replicons in mammalian cells. The latent origin of plasmid replication (oriP), when supplied with the viral Epstein-Barr Nuclear Antigen 1 (EBNA1) in trans, provides efficient duplication, partitioning and maintenance of plasmids bearing it. In this review, we detail what is known about the viral cis and trans elements required for plasmid replication. In addition, we describe how the cellular factors that EBV usurps are used to complement the functions of the viral constituents. Finally, we propose a model for the sequential assembly of an EBNA1-dependent origin of DNA synthesis into a pre-Replicative Complex (pre-RC), which functions by making use only of cellular enzymatic activities to carry out the replication of the viral plasmid.