Generation of Neutralizing Human Monoclonal Antibodies against Parvovirus B19 Proteins

ABSTRACT Infections caused by human parvovirus B19 are known to be controlled mainly by neutralizing antibodies. To analyze the immune reaction against parvovirus B19 proteins, four cell lines secreting human immunoglobulin G monoclonal antibodies (MAbs) were generated from two healthy donors and one human immunodeficiency virus type 1-seropositive individual with high serum titers against parvovirus. One MAb is specific for nonstructural protein NS1 (MAb 1424), two MAbs are specific for the unique region of minor capsid protein VP1 (MAbs 1418-1 and 1418-16), and one MAb is directed to major capsid protein VP2 (MAb 860-55D). Two MAbs, 1418-1 and 1418-16, which were generated from the same individual have identity in the cDNA sequences encoding the variable domains, with the exception of four base pairs resulting in only one amino acid change in the light chain. The NS1- and VP1-specific MAbs interact with linear epitopes, whereas the recognized epitope in VP2 is conformational. The MAbs specific for the structural proteins display strong virus-neutralizing activity. The VP1- and VP2-specific MAbs have the capacity to neutralize 50% of infectious parvovirus B19 in vitro at 0.08 and 0.73 μg/ml, respectively, demonstrating the importance of such antibodies in the clearance of B19 viremia. The NS1-specific MAb mediated weak neutralizing activity and required 47.7 μg/ml for 50% neutralization. The human MAbs with potent neutralizing activity could be used for immunotherapy of chronically B19 virus-infected individuals and acutely infected pregnant women. Furthermore, the knowledge gained regarding epitopes which induce strongly neutralizing antibodies may be important for vaccine development.

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The VP1u of Human Parvovirus B19: A Multifunctional Capsid Protein with Biotechnological Applications

Viruses ◽

10.3390/v12121463 ◽

2020 ◽

Vol 12 (12) ◽

pp. 1463

Author(s):

Carlos Ros ◽

Jan Bieri ◽

Remo Leisi

Keyword(s):

Capsid Protein ◽

Neutralizing Antibodies ◽

Parvovirus B19 ◽

Membrane Receptors ◽

Endosomal Escape ◽

Erythroid Cell ◽

Human Parvovirus B19 ◽

Unique Region ◽

Differentiation Stage ◽

Viral Protein 1

The viral protein 1 unique region (VP1u) of human parvovirus B19 (B19V) is a multifunctional capsid protein with essential roles in virus tropism, uptake, and subcellular trafficking. These functions reside on hidden protein domains, which become accessible upon interaction with cell membrane receptors. A receptor-binding domain (RBD) in VP1u is responsible for the specific targeting and uptake of the virus exclusively into cells of the erythroid lineage in the bone marrow. A phospholipase A2 domain promotes the endosomal escape of the incoming virus. The VP1u is also the immunodominant region of the capsid as it is the target of neutralizing antibodies. For all these reasons, the VP1u has raised great interest in antiviral research and vaccinology. Besides the essential functions in B19V infection, the remarkable erythroid specificity of the VP1u makes it a unique erythroid cell surface biomarker. Moreover, the demonstrated capacity of the VP1u to deliver diverse cargo specifically to cells around the proerythroblast differentiation stage, including erythroleukemic cells, offers novel therapeutic opportunities for erythroid-specific drug delivery. In this review, we focus on the multifunctional role of the VP1u in B19V infection and explore its potential in diagnostics and erythroid-specific therapeutics.

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The P. falciparum CSP repeat region contains three distinct epitopes required for protection by antibodies in vivo

PLoS Pathogens ◽

10.1371/journal.ppat.1010042 ◽

2021 ◽

Vol 17 (11) ◽

pp. e1010042

Author(s):

Yevel Flores-Garcia ◽

Lawrence T. Wang ◽

Minah Park ◽

Beejan Asady ◽

Azza H. Idris ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Monoclonal Antibodies ◽

Malaria Infection ◽

Vaccine Development ◽

Circumsporozoite Protein ◽

Repeat Region ◽

Human Mabs ◽

Necessary And Sufficient

Rare and potent monoclonal antibodies (mAbs) against the Plasmodium falciparum (Pf) circumsporozoite protein (CSP) on infective sporozoites (SPZ) preferentially bind the PfCSP junctional tetrapeptide NPDP or NVDP minor repeats while cross-reacting with NANP major repeats in vitro. The extent to which each of these epitopes is required for protection in vivo is unknown. Here, we assessed whether junction-, minor repeat- and major repeat-preferring human mAbs (CIS43, L9 and 317 respectively) bound and protected against in vivo challenge with transgenic P. berghei (Pb) SPZ expressing either PfCSP with the junction and minor repeats knocked out (KO), or PbCSP with the junction and minor repeats knocked in (KI). In vivo protection studies showed that the junction and minor repeats are necessary and sufficient for CIS43 and L9 to neutralize KO and KI SPZ, respectively. In contrast, 317 required major repeats for in vivo protection. These data establish that human mAbs can prevent malaria infection by targeting three different protective epitopes (NPDP, NVDP, NANP) in the PfCSP repeat region. This report will inform vaccine development and the use of mAbs to passively prevent malaria.

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The VP1-unique region of parvovirus B19: amino acid variability and antigenic stability

Journal of General Virology ◽

10.1099/0022-1317-82-1-191 ◽

2001 ◽

Vol 82 (1) ◽

pp. 191-199 ◽

Cited By ~ 33

Author(s):

Simone Dorsch ◽

Bärbel Kaufmann ◽

Uwe Schaible ◽

Elke Prohaska ◽

Hans Wolf ◽

...

Keyword(s):

Amino Acid ◽

Amniotic Fluid ◽

Peripheral Blood ◽

Neutralizing Antibodies ◽

Parvovirus B19 ◽

Recombinant Baculovirus ◽

Amino Acid Sequences ◽

Human Mabs ◽

Unique Region ◽

Amino Acid Variability

The unique region of structural protein VP1 of parvovirus B19 (erythrovirus B19) is important for eliciting neutralizing antibodies that are responsible for eliminating the virus from the peripheral blood and for inducing lifelong immunity. Neutralizing human MAbs bind a conformationally defined epitope spanning VP1 residues 30–42. The DNA sequence encoding the VP1-unique region was determined in parvovirus B19 isolated from peripheral blood and amniotic fluid of nine acutely infected pregnant women, five arthritis patients and two chronically infected children. The amino acid sequences of the VP1-unique region exhibited higher variability in comparison with other B19-specific proteins. To analyse the influence of amino acid variations on antibody binding and protein conformation, two variants of the VP1-unique region were selected and expressed in E. coli as intein-fusion proteins. The selected variants displayed a number of amino acid exchanges in the VP1-unique region and had mutations in the determined epitope and adjacent regions. After purification via affinity chromatography, the dissociation constants K D of VP1-specific human MAbs interacting with the variant antigens and a viral prototype of the VP1-unique region were determined with a quartz crystal microbalance biosensor. A value of 5·4×10−8 M was determined for the prototype isolate pJB; the affinity constants for the variant VP1-unique regions were similar. Comparable values were obtained for interaction of antibodies with non-infectious VP1/VP2 capsids produced by recombinant baculovirus and with B19 virions from amniotic fluid. It is concluded that the conformation of the epitope is unaffected by mutations or the environment of the VP1-unique region in virus capsids.

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Molecular basis of immune evasion by the delta and kappa SARS-CoV-2 variants

10.1101/2021.08.11.455956 ◽

2021 ◽

Author(s):

Matthew McCallum ◽

Alexandra C Walls ◽

Kaitlin R Sprouse ◽

John E Bowen ◽

Laura Rosen ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Immune Evasion ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

Terminal Domain ◽

Structural Framework ◽

Ancestral Virus ◽

Delta Receptor ◽

The Impact

Worldwide SARS-CoV-2 transmission leads to the recurrent emergence of variants, such as the recently described B.1.617.1 (kappa), B.1.617.2 (delta) and B.1.617.2+ (delta+). The B.1.617.2 (delta) variant of concern is causing a new wave of infections in many countries, mostly affecting unvaccinated individuals, and has become globally dominant. We show that these variants dampen the in vitro potency of vaccine-elicited serum neutralizing antibodies and provide a structural framework for describing the impact of individual mutations on immune evasion. Mutations in the B.1.617.1 (kappa) and B.1.617.2 (delta) spike glycoproteins abrogate recognition by several monoclonal antibodies via alteration of key antigenic sites, including an unexpected remodeling of the B.1.617.2 (delta) N-terminal domain. The binding affinity of the B.1.617.1 (kappa) and B.1.617.2 (delta) receptor-binding domain for ACE2 is comparable to the ancestral virus whereas B.1.617.2+ (delta+) exhibits markedly reduced affinity. We describe a previously uncharacterized class of N-terminal domain-directed human neutralizing monoclonal antibodies cross-reacting with several variants of concern, revealing a possible target for vaccine development.

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Non-neutralizing antibodies targeting the immunogenic regions of HIV-1 envelope reduce mucosal infection and virus burden in humanized mice

PLoS Pathogens ◽

10.1371/journal.ppat.1010183 ◽

2022 ◽

Vol 18 (1) ◽

pp. e1010183

Author(s):

Catarina E. Hioe ◽

Guangming Li ◽

Xiaomei Liu ◽

Ourania Tsahouridis ◽

Xiuting He ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Vaccine Trial ◽

Microbial Pathogens ◽

Humanized Mice ◽

Tier 2 ◽

Human Mabs ◽

Hematopoietic Stem ◽

Neutralizing Activity ◽

Hiv 1

Antibodies are principal immune components elicited by vaccines to induce protection from microbial pathogens. In the Thai RV144 HIV-1 vaccine trial, vaccine efficacy was 31% and the sole primary correlate of reduced risk was shown to be vigorous antibody response targeting the V1V2 region of HIV-1 envelope. Antibodies against V3 also were inversely correlated with infection risk in subsets of vaccinees. Antibodies recognizing these regions, however, do not exhibit potent neutralizing activity. Therefore, we examined the antiviral potential of poorly neutralizing monoclonal antibodies (mAbs) against immunodominant V1V2 and V3 sites by passive administration of human mAbs to humanized mice engrafted with CD34+ hematopoietic stem cells, followed by mucosal challenge with an HIV-1 infectious molecular clone expressing the envelope of a tier 2 resistant HIV-1 strain. Treatment with anti-V1V2 mAb 2158 or anti-V3 mAb 2219 did not prevent infection, but V3 mAb 2219 displayed a superior potency compared to V1V2 mAb 2158 in reducing virus burden. While these mAbs had no or weak neutralizing activity and elicited undetectable levels of antibody-dependent cellular cytotoxicity (ADCC), V3 mAb 2219 displayed a greater capacity to bind virus- and cell-associated HIV-1 envelope and to mediate antibody-dependent cellular phagocytosis (ADCP) and C1q complement binding as compared to V1V2 mAb 2158. Mutations in the Fc region of 2219 diminished these effector activities in vitro and lessened virus control in humanized mice. These results demonstrate the importance of Fc functions other than ADCC for antibodies without potent neutralizing activity.

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Novel Surrogate Neutralizing Assay Supports Parvovirus B19 Vaccine Development for Children with Sickle Cell Disease

Vaccines ◽

10.3390/vaccines9080860 ◽

2021 ◽

Vol 9 (8) ◽

pp. 860

Author(s):

Rhiannon R. Penkert ◽

Sumana Chandramouli ◽

Philip R. Dormitzer ◽

Ethan C. Settembre ◽

Robert E. Sealy ◽

...

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

Parvovirus B19 ◽

Rapid Development ◽

Neutralization Assay ◽

Cross Reactivity ◽

Cell Disease ◽

Unique Region

Children with sickle cell disease (SCD) suffer life-threatening transient aplastic crisis (TAC) when infected with parvovirus B19. In utero, infection of healthy fetuses may result in anemia, hydrops, and death. Unfortunately, although promising vaccine candidates exist, no product has yet been licensed. One barrier to vaccine development has been the lack of a cost-effective, standardized parvovirus B19 neutralization assay. To fill this void, we evaluated the unique region of VP1 (VP1u), which contains prominent targets of neutralizing antibodies. We discovered an antigenic cross-reactivity between VP1 and VP2 that, at first, thwarted the development of a surrogate neutralization assay. We overcame the cross-reactivity by designing a mutated VP1u (VP1uAT) fragment. A new VP1uAT ELISA yielded results well correlated with neutralization (Spearman’s correlation coefficient = 0.581; p = 0.001), superior to results from a standard clinical diagnostic ELISA or an ELISA with virus-like particles. Virus-specific antibodies from children with TAC, measured by the VP1uAT and neutralization assays, but not other assays, gradually increased from days 0 to 120 post-hospitalization. We propose that this novel and technically simple VP1uAT ELISA might now serve as a surrogate for the neutralization assay to support rapid development of a parvovirus B19 vaccine.

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An Exposed Domain in the Severe Acute Respiratory Syndrome Coronavirus Spike Protein Induces Neutralizing Antibodies

Journal of Virology ◽

10.1128/jvi.78.13.7217-7226.2004 ◽

2004 ◽

Vol 78 (13) ◽

pp. 7217-7226 ◽

Cited By ~ 59

Author(s):

Tong Zhou ◽

Hong Wang ◽

Danlin Luo ◽

Thomas Rowe ◽

Zheng Wang ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Monoclonal Antibodies ◽

Severe Acute Respiratory Syndrome ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

Vero Cells ◽

Spike Protein ◽

Protein Fragment

ABSTRACT Exposed epitopes of the spike protein may be recognized by neutralizing antibodies against severe acute respiratory syndrome (SARS) coronavirus (CoV). A protein fragment (S-II) containing predicted epitopes of the spike protein was expressed in Escherichia coli. The properly refolded protein fragment specifically bound to the surface of Vero cells. Monoclonal antibodies raised against this fragment recognized the native spike protein of SARS CoV in both monomeric and trimeric forms. These monoclonal antibodies were capable of blocking S-II attachment to Vero cells and exhibited in vitro antiviral activity. These neutralizing antibodies mapped to epitopes in two peptides, each comprising 20 amino acids. Thus, this region of the spike protein might be a target for generation of therapeutic neutralizing antibodies against SARS CoV and for vaccine development to elicit protective humoral immunity.

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Effects of Size and Surface Properties of Nanodiamonds on the Immunogenicity of Plant-Based H5 Protein of A/H5N1 Virus in Mice

Nanomaterials ◽

10.3390/nano11061597 ◽

2021 ◽

Vol 11 (6) ◽

pp. 1597

Author(s):

Thuong Thi Ho ◽

Van Thi Pham ◽

Tra Thi Nguyen ◽

Vy Thai Trinh ◽

Tram Vi ◽

...

Keyword(s):

High Pressure ◽

Neutralizing Antibodies ◽

H5n1 Virus ◽

Vaccine Development ◽

Particle Characterization ◽

Innovative Strategy ◽

Specific Igg ◽

Positively Charged

Nanodiamond (ND) has recently emerged as a potential nanomaterial for nanovaccine development. Here, a plant-based haemagglutinin protein (H5.c2) of A/H5N1 virus was conjugated with detonation NDs (DND) of 3.7 nm in diameter (ND4), and high-pressure and high-temperature (HPHT) oxidative NDs of ~40–70 nm (ND40) and ~100–250 nm (ND100) in diameter. Our results revealed that the surface charge, but not the size of NDs, is crucial to the protein conjugation, as well as the in vitro and in vivo behaviors of H5.c2:ND conjugates. Positively charged ND4 does not effectively form stable conjugates with H5.c2, and has no impact on the immunogenicity of the protein both in vitro and in vivo. In contrast, the negatively oxidized NDs (ND40 and ND100) are excellent protein antigen carriers. When compared to free H5.c2, H5.c2:ND40, and H5.c2:ND100 conjugates are highly immunogenic with hemagglutination titers that are both 16 times higher than that of the free H5.c2 protein. Notably, H5.c2:ND40 and H5.c2:ND100 conjugates induce over 3-folds stronger production of both H5.c2-specific-IgG and neutralizing antibodies against A/H5N1 than free H5.c2 in mice. These findings support the innovative strategy of using negatively oxidized ND particles as novel antigen carriers for vaccine development, while also highlighting the importance of particle characterization before use.

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The Effect of a Unique Region of Parvovirus B19 Capsid Protein VP1 on Endothelial Cells

Biomolecules ◽

10.3390/biom11040606 ◽

2021 ◽

Vol 11 (4) ◽

pp. 606

Author(s):

Ieva Rinkūnaitė ◽

Egidijus Šimoliūnas ◽

Daiva Bironaitė ◽

Rasa Rutkienė ◽

Virginija Bukelskienė ◽

...

Keyword(s):

Endothelial Cells ◽

Umbilical Vein ◽

Parvovirus B19 ◽

Acute Infection ◽

Endothelial Damage ◽

Precursor Cells ◽

Unique Region ◽

Erythroid Precursor ◽

Active Replication

Parvovirus B19 (B19V) is a widespread human pathogen possessing a high tropism for erythroid precursor cells. However, the persistence or active replication of B19V in endothelial cells (EC) has been detected in diverse human pathologies. The VP1 unique region (VP1u) of the viral capsid has been reported to act as a major determinant of viral tropism for erythroid precursor cells. Nevertheless, the interaction of VP1u with EC has not been studied. We demonstrate that recombinant VP1u is efficiently internalized by rats’ pulmonary trunk blood vessel-derived EC in vitro compared to the human umbilical vein EC line. The exposure to VP1u was not acutely cytotoxic to either human- or rat-derived ECs, but led to the upregulation of cellular stress signaling-related pathways. Our data suggest that high levels of circulating B19V during acute infection can cause endothelial damage, even without active replication or direct internalization into the cells.

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Near-germline human monoclonal antibodies neutralize and protect against multiple arthritogenic alphaviruses

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2100104118 ◽

2021 ◽

Vol 118 (37) ◽

pp. e2100104118

Author(s):

Ryan J. Malonis ◽

James T. Earnest ◽

Arthur S. Kim ◽

Matthew Angeliadis ◽

Frederick W. Holtsberg ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Neutralizing Antibodies ◽

Natural Infection ◽

Human Mabs ◽

Isolation And Characterization ◽

Neutralizing Capacity ◽

Rheumatological Disease ◽

Mayaro Virus ◽

Alphavirus Infection ◽

Globally Distributed

Arthritogenic alphaviruses are globally distributed, mosquito-transmitted viruses that cause rheumatological disease in humans and include Chikungunya virus (CHIKV), Mayaro virus (MAYV), and others. Although serological evidence suggests that some antibody-mediated heterologous immunity may be afforded by alphavirus infection, the extent to which broadly neutralizing antibodies that protect against multiple arthritogenic alphaviruses are elicited during natural infection remains unknown. Here, we describe the isolation and characterization of MAYV-reactive alphavirus monoclonal antibodies (mAbs) from a CHIKV-convalescent donor. We characterized 33 human mAbs that cross-reacted with CHIKV and MAYV and engaged multiple epitopes on the E1 and E2 glycoproteins. We identified five mAbs that target distinct regions of the B domain of E2 and potently neutralize multiple alphaviruses with differential breadth of inhibition. These broadly neutralizing mAbs (bNAbs) contain few somatic mutations and inferred germline–revertants retained neutralizing capacity. Two bNAbs, DC2.M16 and DC2.M357, protected against both CHIKV- and MAYV-induced musculoskeletal disease in mice. These findings enhance our understanding of the cross-reactive and cross-protective antibody response to human alphavirus infections.

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