Isolation of the first human monoclonal antibodies to Sosuga virus
ELISA assays with VLPs would be similar to authentic viruses, but known methods for producing VLPs for other rubulaviruses require the use of matrix (M) and nucleoprotein (N) structural proteins (66). The presence of these structural proteins in the screen can lead to the isolation of SOSV-specific B cells that are not reactive to any of the glycoproteins. Cell surface display allows wild-type transmembrane proteins such as SOSV glycoproteins to be expressed and displayed on the cell surface.
To confirm expression of the SOSV proteins, both Gen1 and Gen2 constructs were transfected into adherent cells (HEp-2 or Vero) to check for syncytia formation in single or co-transfected wells. Tags are added to the carboxy terminus of the F protein, while tags are added to the amino terminus of the HN proteins. Finally, the intensity of the secondary staining was used to determine which cells were labeled with antibodies.
It is important to note that the number of isolated mAbs reactive to each glycoprotein does not reflect the prevalence of mAbs in the donor. Second, at the beginning of this project I was thinking closer to working on RSV than rubulaviruses like mumps. Because RSV F is a major target for vaccines and therapeutic mAbs ( 72 , 74 ), greater attention was paid to SOSV F-specific B cells at the beginning of this project.
Cells were then washed with DPBS before being stained with 50 L/well DAPI (4,6-diamidino-2-phenylindole, dihydrochloride) (Invitrogen, cat. #D1306) diluted to 5 M in DPBS for 15 min . Ab IgG proteins in supernatants of cloned hybridoma cell lines were prepared by washing cells from T-75 flasks in serum-free Hybridoma-SFM Medium (Thermo Fisher Scientific, cat.. 80240M) with the mAb-secreting lines in Hybridoma-SFM medium. Plasmids encoding the recombinant mAbs were expressed using the ExpiCHO expression system (Thermo Fisher Scientific, cat. # A29130).
Characterization of anti-SOSV recombinant monoclonal antibodies
Guillaume Stewart-Jones formerly of the Vaccine Research Center (VRC), of the National Institute of Allergy and Infectious Diseases (NIAID), of the National. Only the glycoprotein specificity of the anti-SOSV mAbs was known from the cell surface display screening. In this chapter, I will discuss the protein domain/conformational specificity of the anti-SOSV mAbs, their competition binding groups, and cross-reactivity with other members of Rubulavirinae.
Guillaume Stewart-Jones of the Vaccine Research Center (VRC) designed soluble forms of the SOSV F protein using similar work for parainfluenza viruses (95). ELISA assays were used to assess the binding of mAbs to the HNhead protein and the tetrameric/dimeric HNecto protein in an ELISA. The half-maximum effective concentration (EC50) values for the binding of rmAbs to soluble HN proteins ranged from 22 to 92 ng/mL (Table 3.1).
Relative EC50 values were obtained for each of the six anti-HN SOSV mAbs against HNecto and HNhead. Data were converted to percent binding relative to maximal noncompetitive binding of the second antibody (lacking a primary mAb). Relative half-maximal effective concentration (EC50) values for binding of rmAbs are summarized in (Table 3.2).
EC50 values were obtained for each of 18 anti-F rmAbs against pre-fusion (preF-tHS) and post-fusion (postF-tHS) F proteins. Data were converted to percent binding relative to the maximum uncompetitive binding of the second antibody (no primary mAb). Cross-reactivity against some orthorubulaviruses (106) or between orthorubulaviruses and respiroviruses (107) has been observed with murine mAbs.
The RSOSV-24 Fab binds to the top of one of the globular head domains, somewhat close to the dimer interface (Figure 3.11).
In conclusion, the work in this chapter provided further insight into the epitope specificities of the panel of anti-SOSV mAbs. Here, I sought to determine whether any of 24 anti-SOSV rmAbs could neutralize a recombinant or. Some of the anti-SOSV mabs had ultrapower. neutralizing activity with IC50 values <1 ng/mL. Neutralization assay of mAb SOSV against live virus.
Many of the control conditions also did not stain, suggesting a possible problem with the SOSV staining. Half of the plate was fixed and stained to determine the amount of SOSV protein expression on the cell surface. rVSV-SOSVT2A-eGFP virus was more cytopathic and killed many of the cells in the 10-1 wells.
However, my collaborators and I suspect that the cytoplasmic tails of SOSV proteins are potentially to blame. Some of the wells that were not inoculated with virus were transfected with SOSV (SOSV F-WT, SOSV HN-WT, or SOSV F+HN WT) or a reporter plasmid (eGFP) to serve as staining controls. The other half of the plate was fixed without permeability as the method in Chapter III.
Recombinant VSV expressing GFP instead of the VSV glycoprotein (rVSVG-GFP) (118) was purchased from Kerafast (cat. # EH1019-PM). One of the broader questions in this project that I was interested in was the possibility of Some of the initial studies for this showed that syncytia could be measured and inhibited.
This allowed us to generate mAb lineage phylogenies for the 9 anti-SOSV mAb heavy chains. In addition, many of these antibodies have been shown to be quite potent neutralizers, with some anti-HN rmAbs being ultra-potent.
Neutralization of SOSV by human monoclonal antibodies
From Chapter III we know the entire panel of protein specificity, conformation/domain specificity and competition binding moieties of anti-SOSV rmAbs. Here in Chapter IV, I will discuss the neutralization capabilities of the anti-SOSV rmAbs, as well as BLS-2 systems for assessing neutralization and the difficulties I encountered with these systems for SOSV. One of the challenges of this dissertation project is that live SOSV is currently considered a BSL-3 virus (65) that exceeds the biosafety levels of our laboratory at Vanderbilt.
One possible solution to this problem has been the construction of pseudoviruses—non-SOSV backbone viruses that display (pseudotyped) SOSV F and HN proteins. Pseudotyping enables the generation of viral particles expressing the envelope proteins of BSL-3/BSL-4 target viruses under BSL-2 conditions. VSV is a single-stranded, nonsegmented, negative-sense, enveloped RNA virus of the Rhabdoviridae family (117).
As a member of Retroviridae (genus Lentivirus), HIV-1 is an enveloped, positive-stranded RNA virus with two copies of the viral. The HIV-1 env gene encodes the polyprotein gp160 which is proteolytically cleaved in the RBP, gp120, which is on the outer surface of the virion and gp41 which is a transmembrane protein in the lipid bilayer that forms a complex with gp120 and acts as the fusion protein (139, 140). For simplicity, the HIV-1 envelope glycoprotein complex (gp120 and gp41) will be called HIV-Env for the remainder of this chapter.
The paramyxoviruses that have been well studied for pseudotyping with either the VSV or lentivirus systems are henipaviruses, morbilliviruses, or respiroviruses, all members within genera of the Orthoparamyxovirinae subfamily (2, 21).
Albariño from the Viral Special Pathogens Branch, Centers for Disease Control and Prevention (CDC), so that neutralization assays would be performed as a single-blind study. Almost all of the rSOSV mAbs were neutralizing, only the postfusion-specific anti-F rmAb was unable to neutralize the virus to an appreciable amount. Of the panel of anti-F mAbs, the prefusion-specific ones tended to have lower IC50 values than the mAbs that bind pre- and postfusion rmAbs.
SOSV rmAbs capable of binding by Western blotting would be identified by costaining the human anti-SOSV rmAbs with the mouse anti-FLAG. Both anti-SOSV rmAbs bound to their respective antigens regardless of the presence of the FLAG tag (Figure 4.4), indicating that these antibodies could be. Blue arrows point to selected bands of the molecular weight marker, as well as the PCNA and SOSV glycoproteins.
Arrows indicate select bands of the molecular weight markers, which are in every other lane. The rVSV-SOSVWT-eGFP virus had a very low titer, as <2% of the cells were infected even in the wells with the highest concentration of virus, but the virus did have cell surface-expressed SOSV proteins ( Table 4.2). 24 hours post-inoculation, half of the plates were fixed while the other half were fixed and permeabilized to determine the amount of surface versus total SOSV protein expression.
All HIV-1 systems failed to produce SOSV pseudoviruses, but the VSV G protein could be pseudotyped (Figure 4.7).
I was hoping there would be much broader reactivity, but most of the cross-reactivity was between SOSV and its close relative (Tuhoko virus 3). The age of the SOSV donor at the time of infection correlates well with the waning immune response to mumps virus observed with MMR vaccinations in young adults. Only one of the antibodies could be considered non-neutralizing (rSOSV-85, postfusion specific), and all neutralizing antibodies had IC50 values.
The 3D reconstruction for rSOSV-10 suggests binding around the top of the trimer, which may lock the F protein in the prefusion conformation. To address this, I had generated both chimeric versions of the F and HN protein with PIV5 or VSV cytoplasmic tails. A construct of the SOSV HN stem (residues 1-125) has already been prepared, expressed and shown to induce syncytia in transfected monolayers.
Since the SOSV antibodies performed well during in vitro neutralization tests, it would be interesting to see if any of the mAbs could be used as prophylaxis in a small animal model. I found the whole case study of the SOSV donor becoming infected fascinating, and I'm glad I was able to study such an interesting virus. Spring-loaded model revisited: Paramyxovirus fusion requires involvement of a receptor-binding protein beyond the initial triggering of the fusion protein.
An electron microscopic study of temperate and virulent interactions between parainfluenza virus SV5 and cells. Development of Twin-Strep-Tag and its use for purification of recombinant proteins from cell culture supernatants. The specificity of the parainfluenza virus hemagglutinin-neuraminidase protein fusion protein is not defined solely by the primary structure of its stalk domain.
Conclusions and future directions
