scholarly journals A plant-biotechnology approach for producing highly potent anti-HIV antibodies for antiretroviral therapy consideration

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Advaita Acarya Singh ◽  
Priyen Pillay ◽  
Lusisizwe Kwezi ◽  
Tsepo Lebiletsa Tsekoa
Keyword(s):  
Posttranslational Modifications ◽  
Nicotiana Benthamiana ◽  
Neutralizing Antibodies ◽  
Production Systems ◽  
Plant Biotechnology ◽  
Variable Region ◽  
Short Review ◽  
Gender Inequalities ◽  
In Planta ◽  
Exposure Prophylaxis

AbstractDespite a reduction in global HIV prevalence the development of a pipeline of new therapeutics or pre-exposure prophylaxis to control the HIV/AIDS epidemic are of high priority. Antibody-based therapies offer several advantages and have been shown to prevent HIV-infection. Plant-based production is efficient for several biologics, including antibodies. We provide a short review on the work by Singh et al., 2020 who demonstrated the transient production of potent CAP256-VRC26 broadly neutralizing antibodies. These antibodies have engineered posttranslational modifications, namely N-glycosylation in the fragment crystallizable region and O-sulfation of tyrosine residues in the complementary-determining region H3 loop. The glycoengineered Nicotiana benthamiana mutant (ΔXTFT) was used, with glycosylating structures lacking β1,2-xylose and/or α1,3-fucose residues, which is critical for enhanced effector activity. The CAP256-VRC26 antibody lineage targets the first and second variable region of the HIV-1 gp120 envelope glycoprotein. The high potency of this lineage is mediated by a protruding O-sulfated tyrosine in the CDR H3 loop. Nicotiana benthamiana lacks human tyrosyl protein sulfotransferase 1, the enzyme responsible for tyrosine O-sulfation. The transient coexpression of the CAP256-VRC26 antibodies with tyrosyl protein sulfotransferase 1 in planta had restored the efficacy of these antibodies through the incorporation of the O-sulfation modification. This approach demonstrates the strategic incorporation of posttranslational modifications in production systems, which may have not been previously considered. These plant-produced CAP256-VRC26 antibodies have therapeutic as well as topical and systemic pre-exposure prophylaxis potential in enabling the empowerment of young girls and women given that gender inequalities remain a major driver of the epidemic.

scholarly journals Simple and efficient heterologous expression of necrosis-inducing effectors using the model plant Nicotiana benthamiana

2021 ◽  
Author(s):  
Bayantes Dagvadorj ◽  
Peter Solomon
Keyword(s):  
Heterologous Expression ◽  
High Throughput ◽  
Posttranslational Modifications ◽  
Nicotiana Benthamiana ◽  
Fungal Pathogens ◽  
Host Plants ◽  
Plant Pathogens ◽  
Expression Systems ◽  
In Planta ◽  
Model Plant

Plant fungal pathogens cause devastating diseases on cereal plants and threaten global food security. During infection, these pathogens secrete proteinaceous effectors that promote disease. Some of these effectors from necrotrophic plant pathogens induce a cell death response (necrosis), which facilitates pathogen growth in planta. Characterisation of these effectors typically requires heterologous expression and microbial expression systems such as bacteria and yeast are the predominantly used. However, microbial expression systems often require optimization for any given effector and are, in general, not suitable for effectors involving cysteine bridges and posttranslational modifications for activity. Here, we describe a simple and efficient method for expressing such effectors in the model plant Nicotiana benthamiana. Briefly, an effector protein is transiently expressed and secreted into the apoplast of N. benthamiana by Agrobacterium-mediated infiltration. Two-to-three days subsequent to agroinfiltration, the apoplast from the infiltrated leaves is extracted and can be directly used for phenotyping on host plants. The efficacy of this approach was demonstrated by expressing the ToxA, Tox3 and Tox1 necrosis-inducing effectors from Parastagonospora nodorum. All three effectors produced in N. benthamiana were capable of inducing necrosis in wheat lines, and two of three showed visible bands on Coomassie-stained gel. These data suggest that N. benthamiana-agroinfiltration system is a feasible tool to obtain fungal effectors, especially those that require disulfide bonds and posttranslational modifications. Furthermore, due to the low number of proteins typically observed in the apoplast (compared to intracellular), this simple and high-throughput approach circumvents the requirement to lyse cells and further purify the target proteins that is required in other heterologous systems. Because of its simplicity and potential for high-throughput, this method is highly amenable to the phenotyping of candidate protein effectors on host plants.

scholarly journals Screening of potent neutralizing antibodies against SARS-CoV-2 using convalescent patients-derived phage-display libraries

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Yongbing Pan ◽  
Jianhui Du ◽  
Jia Liu ◽  
Hai Wu ◽  
Fang Gui ◽  
...  
Keyword(s):  
Phage Display ◽  
Neutralizing Antibodies ◽  
Variable Region ◽  
Neutralizing Antibody ◽  
Chain Gene ◽  
Prophylactic Efficacy ◽  
Heavy Chains ◽  
Effective Interventions ◽  
Phage Display Libraries

AbstractAs the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to threaten public health worldwide, the development of effective interventions is urgently needed. Neutralizing antibodies (nAbs) have great potential for the prevention and treatment of SARS-CoV-2 infection. In this study, ten nAbs were isolated from two phage-display immune libraries constructed from the pooled PBMCs of eight COVID-19 convalescent patients. Eight of them, consisting of heavy chains encoded by the immunoglobulin heavy-chain gene-variable region (IGHV)3-66 or IGHV3-53 genes, recognized the same epitope on the receptor-binding domain (RBD), while the remaining two bound to different epitopes. Among the ten antibodies, 2B11 exhibited the highest affinity and neutralization potency against the original wild-type (WT) SARS-CoV-2 virus (KD = 4.76 nM for the S1 protein, IC50 = 6 ng/mL for pseudoviruses, and IC50 = 1 ng/mL for authentic viruses), and potent neutralizing ability against B.1.1.7 pseudoviruses. Furthermore, 1E10, targeting a distinct epitope on RBD, exhibited different neutralization efficiency against WT SARS-CoV-2 and its variants B.1.1.7, B.1.351, and P.1. The crystal structure of the 2B11–RBD complexes revealed that the epitope of 2B11 highly overlaps with the ACE2-binding site. The in vivo experiment of 2B11 using AdV5-hACE2-transduced mice showed encouraging therapeutic and prophylactic efficacy against SARS-CoV-2. Taken together, our results suggest that the highly potent SARS-CoV-2-neutralizing antibody, 2B11, could be used against the WT SARS-CoV-2 and B.1.1.7 variant, or in combination with a different epitope-targeted neutralizing antibody, such as 1E10, against SARS-CoV-2 variants.

scholarly journals Characterization of Neutralizing Antibody Responses Elicited by Clade A Envelope Immunogens Derived from Early Transmitted Viruses

2008 ◽  
Vol 82 (12) ◽  
pp. 5912-5921 ◽  
Author(s):  
Zane Kraft ◽  
Katharine Strouss ◽  
William F. Sutton ◽  
Brad Cleveland ◽  
For Yue Tso ◽  
...  
Keyword(s):  
Chronic Infection ◽  
Neutralizing Antibodies ◽  
Acute Infection ◽  
Variable Region ◽  
Neutralizing Antibody ◽  
Antibody Responses ◽  
Virus Envelope ◽  
Variable Regions ◽  
Clade B ◽  
Region Length

ABSTRACT The vast majority of studies with candidate immunogens based on the human immunodeficiency virus envelope (Env) have been conducted with Env proteins derived from clade B viruses isolated during chronic infection. Whether non-clade B Env protein immunogens will elicit antibodies with epitope specificities that are similar to those of antibodies elicited by clade B Envs and whether the antibodies elicited by Envs derived from early transmitted viruses will be similar to those elicited by Envs derived from viruses isolated during chronic infection are currently unknown. Here we performed immunizations with four clade A Envs, cloned directly from the peripheral blood of infected individuals during acute infection, which differed in lengths and extents of glycosylation. The antibody responses elicited by these four Envs were compared to each other and to those elicited by a well-characterized clade B Env immunogen derived from the SF162 virus, which was isolated during chronic infection. Only one clade A Env, the one with the fewer glycosylation sites, elicited homologous neutralizing antibodies (NAbs); these did not target the V1, V2, or V3 regions. In contrast, all four clade A Envs elicited anti-V3 NAbs against “easy-to-neutralize” clade B and clade A isolates, irrespective of the variable region length and extent of glycosylation of the Env used as an immunogen. These anti-V3 NAbs did not access their epitopes on homologous and heterologous clade A, or B, neutralization-resistant viruses. The length and extent of glycosylation of the variable regions on the clade A Env immunogens tested did not affect the breadth of the elicited NAbs. Our data also indicate that the development of cross-reactive NAbs against clade A viruses faces similar hurdles to the development of cross-reactive anti-clade B NAbs.

A vector system for fast-forward studies of the HOPZ-ACTIVATED RESISTANCE1 (ZAR1) resistosome in the model plant Nicotiana benthamiana

2021 ◽  
Author(s):  
Adeline Harant ◽  
Hsuan Pai ◽  
Toshiyuki Sakai ◽  
Sophien Kamoun ◽  
Hiroaki Adachi
Keyword(s):  
Nicotiana Benthamiana ◽  
Cell Biology ◽  
Plant Immunity ◽  
Transient Gene Expression ◽  
Coiled Coil ◽  
Experimental System ◽  
Vector System ◽  
In Planta ◽  
Functional Studies

Abstract Nicotiana benthamiana has emerged as a complementary experimental system to Arabidopsis thaliana. It enables fast-forward in vivo analyses primarily through transient gene expression and is particularly popular in the study of plant immunity. Recently, our understanding of nucleotide-binding leucine-rich repeat (NLR) plant immune receptors has greatly advanced following the discovery of the Arabidopsis HOPZ-ACTIVATED RESISTANCE1 (ZAR1) resistosome. Here, we describe a vector system of 72 plasmids that enables functional studies of the ZAR1 resistosome in N. benthamiana. We showed that ZAR1 stands out among the coiled coil class of NLRs (CC-NLRs) for being highly conserved across distantly related dicot plant species and confirmed NbZAR1 as the N. benthamiana ortholog of Arabidopsis ZAR1. Effector-activated and autoactive NbZAR1 trigger the cell death response in N. benthamiana and this activity is dependent on a functional N-terminal α1 helix. C-terminally tagged NbZAR1 remains functional in N. benthamiana, thus enabling cell biology and biochemical studies in this plant system. We conclude that the NbZAR1 open source pZA plasmid collection forms an additional experimental system to Arabidopsis for in planta resistosome studies.

scholarly journals Evidence for Functional Diversification Within a Fungal NEP1-Like Protein Family

2013 ◽  
Vol 26 (3) ◽  
pp. 278-286 ◽  
Author(s):  
Parthasarathy Santhanam ◽  
H. Peter van Esse ◽  
Isabell Albert ◽  
Luigi Faino ◽  
Thorsten Nürnberger ◽  
...  
Keyword(s):  
Gene Family ◽  
Vegetative Growth ◽  
Nicotiana Benthamiana ◽  
Family Members ◽  
Aerial Mycelium ◽  
Vascular Wilt ◽  
In Planta ◽  
Functional Diversification ◽  
Targeted Deletion ◽  
Genes Encoding

In this study, we functionally analyzed the gene family encoding necrosis- and ethylene-inducing-like proteins (NLP) of the vascular wilt pathogen Verticillium dahliae. We show that the composition of the NLP gene family varies little among V. dahliae isolates. The cytotoxic activity of NLP family members of a tomato-pathogenic V. dahliae strain was determined, demonstrating that only two of the seven NLP induced plant cell death. The genes encoding these cytotoxic NLP were found to be induced in V. dahliae upon colonization of tomato. Interestingly, targeted deletion of either of the two genes in V. dahliae significantly compromised virulence on tomato as well as on Arabidopsis plants, whereas deletion of only one of the two genes affected virulence on Nicotiana benthamiana. This could be attributed to differential induction of the two NLP genes in V. dahliae upon N. benthamiana colonization, revealing that the in planta induction of NLP genes varies between plant hosts. Intriguingly, one of the NLP genes appears to also affect vegetative growth and conidiospore production, because the corresponding deletion strain produced significantly fewer conidiospores and developed extensive aerial mycelium. In conclusion, we demonstrate that the expanded V. dahliae NLP family shows functional diversification, revealing not only differential cytotoxicity between family members but also that the cytotoxic NLP play a role in vegetative growth and asexual reproduction in addition to their contribution to virulence.

scholarly journals The Glycosylphosphatidylinositol Anchor Biosynthesis Genes GPI12, GAA1, and GPI8 Are Essential for Cell-Wall Integrity and Pathogenicity of the Maize Anthracnose Fungus Colletotrichum graminicola

2016 ◽  
Vol 29 (11) ◽  
pp. 889-901 ◽  
Author(s):  
Ely Oliveira-Garcia ◽  
Holger B. Deising
Keyword(s):  
Cell Wall ◽  
Posttranslational Modifications ◽  
Hyphal Growth ◽  
Transcript Abundance ◽  
Essential Genes ◽  
Colletotrichum Graminicola ◽  
In Planta ◽  
Vegetative Development ◽  
Cell Wall Rigidity ◽  
Gpi Transamidase

Glycosylphosphatidylinositol (GPI) anchoring of proteins is one of the most common posttranslational modifications of proteins in eukaryotic cells and is important for associating proteins with the cell surface. In fungi, GPI-anchored proteins play essential roles in cross-linking of β-glucan cell-wall polymers and cell-wall rigidity. GPI-anchor synthesis is successively performed at the cytoplasmic and the luminal face of the ER membrane and involves approximately 25 proteins. While mutagenesis of auxiliary genes of this pathway suggested roles of GPI-anchored proteins in hyphal growth and virulence, essential genes of this pathway have not been characterized. Taking advantage of RNA interference (RNAi) we analyzed the function of the three essential genes GPI12, GAA1 and GPI8, encoding a cytoplasmic N-acetylglucosaminylphosphatidylinositol deacetylase, a metallo-peptide-synthetase and a cystein protease, the latter two representing catalytic components of the GPI transamidase complex. RNAi strains showed drastic cell-wall defects, resulting in exploding infection cells on the plant surface and severe distortion of in planta–differentiated infection hyphae, including formation of intrahyphal hyphae. Reduction of transcript abundance of the genes analyzed resulted in nonpathogenicity. We show here for the first time that the GPI synthesis genes GPI12, GAA1, and GPI8 are indispensable for vegetative development and pathogenicity of the causal agent of maize anthracnose, Colletotrichum graminicola.

Chapter 14: Prevention: vaccines and immunoglobulins

2021 ◽  
Author(s):  
Eva-Maria Pöllabauer ◽  
Herwig Kollaritsch
Keyword(s):  
Immune Responses ◽  
Neutralizing Antibodies ◽  
Western Europe ◽  
Surrogate Marker ◽  
Booster Vaccination ◽  
Post Exposure Prophylaxis ◽  
Pediatric Formulation ◽  
The One ◽  
Exposure Prophylaxis

Worldwide there are 6 different TBE vaccines – two from Western Europe, three from Russia and one from China. The two western European vaccines and one of the Russian vaccines have an adult and a pediatric formulation. The products names are FSME IMMUN and FSME-IMMUN Junior; Encepur adults and Encepur children, Klesch-E-Vac, EnceVir and EnceVir Neo, Dry lyophilized TBE Moscow and Sen Tai Bao. All TBE vaccines except the one from China have similar but not identical immunization schedules with primary immunization (>3 doses) and regular booster vaccinations. For FSME-IMMUN, Encepur and EnceVir rapid immunization schedules are also licensed. The Chinese vaccine is given with 2 primary doses 2 weeks apart followed by annual boosters. All vaccines induce significant immune responses. In the absence of a formal correlate of protection, the presence of neutralizing antibodies is used as a surrogate marker for protection. Recent clinical studies show long-term seropersistence of TBE antibodies after the first booster vaccination (dose 4) with the two European vaccines. An effectiveness of approximately 99% (years 2000–2006) and 98.7% (years 2000-2011) was calculated for regularly vaccinated persons in Austria, a country with established high vaccination uptake. Whereas in Western Europe post-exposure prophylaxis with immunoglobulins was discontinued in the late 1990s, in the highly endemic regions of Russia it continues to be common practice. Both – FSME-IMMUN and Encepur are well tolerated with a well-established safety profile. TBE-Moscow and EnceVir appear to be somewhat more reactogenic.

scholarly journals Analysis of HIV-1 subtype B third variable region peptide motifs for induction of neutralizing antibodies against HIV-1 primary isolates

Virology ◽  
2006 ◽  
Vol 345 (1) ◽  
pp. 44-55 ◽  
Author(s):  
Barton F. Haynes ◽  
Benjiang Ma ◽  
David C. Montefiori ◽  
Terri Wrin ◽  
Christos J. Petropoulos ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Variable Region ◽  
Peptide Motifs ◽  
Subtype B ◽  
Hiv 1

scholarly journals Geminivirus Replication Protein Impairs SUMO Conjugation of Proliferating Cellular Nuclear Antigen at Two Acceptor Sites

2018 ◽  
Vol 92 (18) ◽  
Author(s):  
Manuel Arroyo-Mateos ◽  
Blanca Sabarit ◽  
Francesca Maio ◽  
Miguel A. Sánchez-Durán ◽  
Tabata Rosas-Díaz ◽  
...  
Keyword(s):  
Dna Repair ◽  
Posttranslational Modifications ◽  
Infected Plant ◽  
Plant Cells ◽  
Nuclear Antigen ◽  
Replication Machinery ◽  
In Planta ◽  
Content Type ◽  
Multifunctional Protein ◽  
Cellular Nuclear Antigen

ABSTRACTGeminiviruses are DNA viruses that replicate in nuclei of infected plant cells using the plant DNA replication machinery, including PCNA (proliferating cellular nuclear antigen), a cofactor that orchestrates genome duplication and maintenance by recruiting crucial players to replication forks. These viruses encode a multifunctional protein, Rep, which is essential for viral replication, induces the accumulation of the host replication machinery, and interacts with several host proteins, including PCNA and the SUMO E2 conjugation enzyme (SCE1). Posttranslational modification of PCNA by ubiquitin or SUMO plays an essential role in the switching of PCNA between interacting partners during DNA metabolism processes (e.g., replication, recombination, and repair, etc.). In yeast, PCNA sumoylation has been associated with DNA repair involving homologous recombination (HR). Previously, we reported that ectopic Rep expression results in very specific changes in the sumoylation pattern of plant cells. In this work, we show, using a reconstituted sumoylation system inEscherichia coli, that tomato PCNA is sumoylated at two residues, K254 and K164, and that coexpression of the geminivirus protein Rep suppresses sumoylation at these lysines. Finally, we confirm that PCNA is sumoylatedin plantaand that Rep also interferes with PCNA sumoylation in plant cells.IMPORTANCESUMO adducts have a key role in regulating the activity of animal and yeast PCNA on DNA repair and replication. Our work demonstrates for the first time that sumoylation of plant PCNA occurs in plant cells and that a plant virus interferes with this modification. This work marks the importance of sumoylation in allowing viral infection and replication in plants. Moreover, it constitutes a prime example of how viral proteins interfere with posttranslational modifications of selected host factors to create a proper environment for infection.

scholarly journals Highly active engineered IgG3 antibodies against SARS-CoV-2

2021 ◽  
Vol 118 (42) ◽  
pp. e2107249118
Author(s):  
Somanath Kallolimath ◽  
Lin Sun ◽  
Roman Palt ◽  
Karin Stiasny ◽  
Patrick Mayrhofer ◽  
...  
Keyword(s):  
Nicotiana Benthamiana ◽  
Vaccine Development ◽  
Binding Activity ◽  
The Other ◽  
Cross Linking ◽  
Antigen Binding ◽  
In Planta ◽  
Antigen Binding Site ◽  
Highly Active ◽  
Viral Surface

Monoclonal antibodies (mAbs) that efficiently neutralize SARS-CoV-2 have been developed at an unprecedented speed. Notwithstanding, there is a vague understanding of the various Ab functions induced beyond antigen binding by the heavy-chain constant domain. To explore the diverse roles of Abs in SARS-CoV-2 immunity, we expressed a SARS-CoV-2 spike protein (SP) binding mAb (H4) in the four IgG subclasses present in human serum (IgG1-4) using glyco-engineered Nicotiana benthamiana plants. All four subclasses, carrying the identical antigen-binding site, were fully assembled in planta and exhibited a largely homogeneous xylose- and fucose-free glycosylation profile. The Ab variants ligated to the SP with an up to fivefold increased binding activity of IgG3. Furthermore, all H4 subtypes were able to neutralize SARS-CoV-2. However, H4-IgG3 exhibited an up to 50-fold superior neutralization potency compared with the other subclasses. Our data point to a strong protective effect of IgG3 Abs in SARS-CoV-2 infection and suggest that superior neutralization might be a consequence of cross-linking the SP on the viral surface. This should be considered in therapy and vaccine development. In addition, we underscore the versatile use of plants for the rapid expression of complex proteins in emergency cases.

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