G1 MAbs Research Articles

KLRG1, Another Opportunity for a Breakthrough in MTCL.

Outcomes in mature T-cell lymphomas remain poor, with previous attempts at developing mAbs compromised by limited efficacy and significant immunocompromise. Anti-killer cell lectin-like receptor G1 mAbs may have greater selectivity and specificity for malignant T cells and avoid the toxicity concerns with previous agents. See related article by Assatova et al., p. 2514.

Validation of an antigenic site targeted by monoclonal antibodies against Puumala virus.

Identification of B-cell epitopes facilitates the development of vaccines, therapeutic antibodies and diagnostic tools. Previously, the binding site of the bank vole monoclonal antibody (mAb) 4G2 against Puumala virus (PUUV, an orthohantavirus in the Hantaviridae family of the Bunyavirales order) was predicted using a combination of methods, including pepscan, phage-display, and site-directed mutagenesis of vesicular stomatitis virus (VSV) particles pseudotyped with Gn and Gc glycoproteins from PUUV. These techniques led to the identification of the neutralization escape mutation F915A. To our surprise, a recent crystal structure of PUUV Gc in complex with Fab 4G2 revealed that residue F915 is distal from epitope of mAb 4G2. To clarify this issue and explore potential explanations for the inconsistency, we designed a mutagenesis experiment to probe the 4G2 epitope, with three PUUV pseudoviruses carrying amino acid changes E725A, S944F, and S946F, located within the structure-based 4G2 epitope on the Gc. These amino acid changes were able to convey neutralization escape from 4G2, and S944F and S946F also conveyed escape from neutralization by human mAb 1C9. Furthermore, our mapping of all the known neutralization evasion sites from hantaviral Gcs onto PUUV Gc revealed that over 60 % of these sites reside within or close to the epitope of mAb 4G2, indicating that this region may represent a crucial area targeted by neutralizing antibodies against PUUV, and to a lesser extent, other hantaviruses. The identification of this site of vulnerability could guide the creation of subunit vaccines against PUUV and other hantaviruses in the future.

Neutralizing antibodies targeting a novel epitope on envelope protein exhibited broad protection against flavivirus without risk of disease enhancement

BackgroundFlavivirus causes many serious public health problems worldwide. However, licensed DENV vaccine has restrictions on its use, and there is currently no approved ZIKV vaccine. Development of a potent and safe flavivirus vaccine is urgently needed. As a previous study revealed the epitope, RCPTQGE, located on the bc loop in the E protein domain II of DENV, in this study, we rationally designed and synthesized a series of peptides based on the sequence of JEV epitope RCPTTGE and DENV/ZIKV epitope RCPTQGE.MethodsImmune sera were generated by immunization with the peptides which were synthesized by using five copies of RCPTTGE or RCPTQGE and named as JEV-NTE and DV/ZV-NTE. Immunogenicity and neutralizing abilities of JEV-NTE or DV/ZV-NTE-immune sera against flavivirus were evaluated by ELISA and neutralization tests, respectively. Protective efficacy in vivo were determined by passive transfer the immune sera into JEV-infected ICR or DENV- and ZIKV-challenged AG129 mice. In vitro and in vivo ADE assays were used to examine whether JEV-NTE or DV/ZV-NTE-immune sera would induce ADE.ResultsPassive immunization with JEV-NTE-immunized sera or DV/ZV-NTE-immunized sera could increase the survival rate or prolong the survival time in JEV-challenged ICR mice and reduce the viremia levels significantly in DENV- or ZIKV-infected AG129 mice. Furthermore, neither JEV -NTE- nor DV/ZV-NTE-immune sera induced antibody-dependent enhancement (ADE) as compared with the control mAb 4G2 both in vitro and in vivo.ConclusionsWe showed for the first time that novel bc loop epitope RCPTQGE located on the amino acids 73 to 79 of DENV/ZIKV E protein could elicit cross-neutralizing antibodies and reduced the viremia level in DENV- and ZIKV-challenged AG129 mice. Our results highlighted that the bc loop epitope could be a promising target for flavivirus vaccine development.

Development and in vitro characterization of a humanized scFv against fungal infections.

The resistance and the birth of new intrinsic and multidrug-resistant pathogenic species like C. auris is creating great concern in the antifungal world. Given the limited drug arsenal and the lack of effectiveness of the available compounds, there is an urgent need for innovative approaches. The murine mAb 2G8 was humanized and engineered in silico to develop a single-chain fragment variable (hscFv) antibody against β-1,3-glucans which was then expressed in E. coli. Among the recombinant proteins developed, a soluble candidate with high stability and affinity was obtained. This selected protein is VL-linker-VH oriented, and it is characterized by the presence of two ubiquitin monomers at the N-terminus and a His tag at the C-terminus. This construct, Ub2-hscFv-His, guaranteed stability, solubility, efficient purification and satisfactory recovery of the recombinant product. HscFv can bind β-1,3-glucans both as coated antigens and on C. auris and C. albicans cells similarly to its murine parental and showed long stability and retention of binding ability when stored at 4°, -20° and -80° C. Furthermore, it was efficient in enhancing the antifungal activity of drugs caspofungin and amphotericin B against C. auris. The use of biological drugs as antifungals is limited; here we present a promising hscFv which has the potential to be useful in combination with currently available antifungal drugs.

195 Development of a fetal porcine model for congenital viral infections in the developing CNS

To develop a porcine model for analysis of the effects of congenital viral infections on CNS and spinal (CSF) derived fetal neural precursor (NPC) and neural progenitor cells. Isolation of NPCs from the subventricular zone (SVZ) of the porcine brain or CSF derived from premature (day 104) and post-term (day 135) Cesarean-delivered piglets were plated in 12-well plates. Nonadherent plates were used to generate neurospheres with self-renewal capacity and for long term passages. After a 2-day incubation for neurosphere formation and removal of apoptotic debris, cells were seeded in various conditions including incubation in (1) 10% FBS, (2) Rock Inhibitor (RI)-specific inhibitor of apoptosis, (3) FBS and RI, and (4) Neurobasal media alone (control). After purification and identification of NPCs, cells were then infected with low MOI of clinically isolated first passage ZIKV HN16, then fixed and stained with Flavivirus-specific antibody (MAb 4G2) followed by FITC labeled secondary antibody staining visualization under fluorescence microscopy. Gene expression studies were also performed from RNA from ZIKV infected and mock infected samples to determine effects on genes related to NPC self-renewal and survival. The brain derived cells cultured for 4 days showed three distinct populations including NSCs, NPCs, and a mixed NSC/NPC population. After 21 days of culture, cells shifted from an NSC population toward later within the NPC lineage. Both brain and CSF cells showed relatively equal permissivity to ZIKV viral infection (Fig.1). Moreover, there was no difference in Vimentin gene expression in ZIKV infected cells, while SOX-2 and Nestin gene expression following ZIKV infection was significantly lower (p<0.05; Fig.2). The findings that Nestin expression is downregulated when comparing brain and CSF cells, particularly after ZIKA infection, is of interest since Nestin is postulated to play a role in axonal growth. This study demonstrated the ability to utilize porcine fetal NPCs in a safe and effective manner for the study of NPC development and effects of viral infections

A quantitative luciferase-based cell–cell fusion assay to measure four-serotype dengue virus E protein-triggered membrane fusion

ABSTRACT The class II membrane fusion induced by flavivirus E proteins is a unique pH-dependent membrane fusion process differently from the class I or III membrane fusion by other enveloped virus proteins. The fusion peptide on the DII of the flavivirus E proteins can insert into the cell membrane as a cell entry process besides the receptor bindings. A traditional assay using C6/36 mosquito cells infected by dengue viruses has been previously reported but did not provide efficient quantitation to measure the virus-triggered membrane fusion. Here we reported the development of a quantitative cell fusion assay for four serotypes of dengue viruses and the recently emerged Zika viruses. We used a pCI-neo vector encoding the prME genes of dengue and Zika viruses and investigated the cell fusion in transfected 293, Vero and CHO cells. Donor cells were co-transfection of the prME genes of dengue and Zika prME gene and T7 RNA polymerase to react with the indicator cells transfected with the luciferase gene under the control of the T7 promoter. Quantification of the virus-induced cell fusion was determined by the luciferase expression levels under a switch of pH from 7.4 to 5.4 in the co-cultured donor and indicator cells. The quantitative luciferase-based assay was applied to measure the anti-fusion activity by two monoclonal antibodies mAb 4G2 and mAb DB42 against dengue virus infections. This assay could quality as a quantitative bioassay for testing the potency of anti-fusion monoclonal antibodies.

Development and characterization of novel anti-C5 monoclonal antibodies capable of inhibiting complement in multiple species.

SummaryOver the last decade there has been an explosion in complement therapies; one‐third of the drugs in the clinic or in development target C5 protein. Eculizumab, a monoclonal antibody (mAb) that binds C5 and blocks its cleavage by the convertase, is the current reference standard treatment for atypical haemolytic uraemic syndrome (aHUS) and paroxysmal nocturnal haemoglobinuria (PNH) and in clinical trials for many other diseases. Here we describe a panel of novel anti‐C5 mAb, including mAb that, like Eculizumab, are efficient inhibitors of complement but, unlike Eculizumab, inhibit across species, including human, rat, rabbit and guinea pig. Several inhibitory anti‐C5 mAb were identified and characterized for C5 binding and lytic inhibitory capacity in comparison to current therapeutic anti‐C5 mAb; three clones, 4G2, 7D4 and 10B6, were selected and further characterized for ligand specificity and affinity and cross‐species inhibitory activity. The mAb 10B6 was human‐specific whereas mAb 4G2 and 7D4 efficiently inhibited lysis by human, rabbit and rat serum, and weakly inhibited guinea pig complement; 7D4 also weakly inhibited mouse complement in vitro The rat C5‐cross‐reactive mAb 4G2, when administered intraperitoneally in a rat model of myasthenia gravis, effectively blocked the disease and protected muscle endplates from destruction. To our knowledge this is the first report of an anti‐C5 function blocking mAb that permits preclinical studies in rats.

Intramuscular Adeno-Associated Virus-Mediated Expression of Monoclonal Antibodies Provides 100% Protection Against Ebola Virus Infection in Mice.

The 2013-2016 West Africa outbreak demonstrated the epidemic potential of Ebola virus and highlighted the need for counter strategies. Monoclonal antibody (mAb)-based therapies hold promise as treatment options for Ebola virus infections. However, production of clinical-grade mAbs is labor intensive, and immunity is short lived. Conversely, adeno-associated virus (AAV)-mediated mAb gene transfer provides the host with a genetic blueprint to manufacture mAbs in vivo, leading to steady release of antibody over many months. Here we demonstrate that AAV-mediated expression of nonneutralizing mAb 5D2 or 7C9 confers 100% protection against mouse-adapted Ebola virus infection, while neutralizing mAb 2G4 was 83% protective. A 2-component cocktail, AAV-2G4/AAV-5D2, provided complete protection when administered 7 days prior to challenge and was partially protective with a 3-day lead time. Finally, AAV-mAb therapies provided sustained protection from challenge 5 months following AAV administration. AAV-mAb may be a viable alternative strategy for vaccination against emerging infectious diseases.

A novel mechanism of antibody-mediated enhancement of flavivirus infection.

Antibody-dependent enhancement of viral infection is a well-described phenomenon that is based on the cellular uptake of infectious virus-antibody complexes following their interaction with Fcγ receptors expressed on myeloid cells. Here we describe a novel mechanism of antibody-mediated enhancement of infection by a flavivirus (tick-borne encephalitis virus) in transformed and primary human cells, which is independent of the presence of Fcγ receptors. Using chemical cross-linking and immunoassays, we demonstrate that the monoclonal antibody (mab) A5, recognizing an epitope at the interface of the dimeric envelope protein E, causes dimer dissociation and leads to the exposure of the fusion loop (FL). Under normal conditions of infection, this process is triggered only after virus uptake by the acidic pH in endosomes, resulting in the initiation of membrane fusion through the interaction of the FL with the endosomal membrane. Analysis of virus binding and cellular infection, together with inhibition by the FL-specific mab 4G2, indicated that the FL, exposed after mab A5- induced dimer-dissociation, mediated attachment of the virus to the plasma membrane also at neutral pH, thereby increasing viral infectivity. Since antibody-induced enhancement of binding was not only observed with cells but also with liposomes, it is likely that increased infection was due to FL-lipid interactions and not to interactions with cellular plasma membrane proteins. The novel mechanism of antibody-induced infection enhancement adds a new facet to the complexity of antibody interactions with flaviviruses and may have implications for yet unresolved effects of polyclonal antibody responses on biological properties of these viruses.

The vaccines and antibodies associated with Als3p for treatment of Candida albicans infections.

Candida albicans is the most common fungal microorganism in healthy individuals, as well as the cause of high mortality infections in high-risk hosts such as immunocompromised patients. Antifungal vaccines and monoclonal antibodies useful for active or passive immunizations have recently generated considerable excitement for the treatment of fungal infections. The cell wall proteins of C. albicans, which are crucial for virulence and pathogenicity, are attractive target antigens. Als3p, a member of the C. albicans agglutinin-like sequence (ALS) family, is a hyphal-specific glycophosphatidylinositol cell wall protein that plays a key role in the interaction with host cells. The abundance of Als3p on the hyphal surface makes it an attractive target. For example, the NDV-3 vaccine, targeted at the N-terminus of Als3p, has entered a preparation of Phase 2 clinical trial. The Als3p-specific antibodies include monoclonal antibodies (MAbs) 3-A5, MAb 113, and scFv3. In addition, MAb C7, MAb 3D9.3 and MAb 2G8, which were supposed to be identifying other targets, have also provided good protection by recognizing Als3p. In this review, we summarize the functions of Als3p and highlight the development of the vaccines and the antibodies that are associated, directly or indirectly, with this protein.

PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST THE AVIAN INFECTIOUS BRONCHITIS VIRUS

The use of monoclonal antibodies (MAbs) has contributed to the diagnosis of avian infectious bronchitis (IB), however their use are still limited because the utilized MAbs are imported, increasing the cost of such methodologies. Therefore, given the relevance of the national poultry production as well as the losses caused by the occurrence of IB in commercial poultry flocks, it is important that Brazil can produce biological supplies to use for diagnostic of IB. Thus, the present work reports the production, and characterization of MAbs against the infectious bronchitis virus (IBV). Two groups of three Balb-c female mice were inoculated with inactivated classic or variant IBV for 10 times, once per week. The splenocytes from mouse with higher serum concentration against IBV antigens were harvested and fused with murine Sp2/O-Ag14 myeloma cells. We generated two hybridomas secreting MAbs anti-IBV (MAb 2G4 and 5A11) from the fusion of myeloma cells and B-lymphocytes from a mouse immunized with the variant IBV. Following the characterization by ELISA and Western Blot, it was observed that MAbs recognize the IBV classic and variant types, showing more intense reactions with IBV variant. The MAbs obtained in this study have potential for use in immunodiagnostic for IB.

Production, Quality Control, Stability and Pharmacotoxicity of a Malaria Vaccine Comprising Three Highly Similar PfAMA1 Protein Molecules to Overcome Antigenic Variation.

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a leading asexual blood stage vaccine candidate for malaria. In preparation for clinical trials, three Diversity Covering (DiCo) PfAMA1 ectodomain proteins, designed to overcome the intrinsic polymorphism that is present in PfAMA1, were produced under Good Manufacturing Practice (GMP) in Pichia pastoris. Using identical methodology, the 3 strains were cultivated in 70-L scale fed-batch fermentations and PfAMA1-DiCos were purified by two chromatography steps, an ultrafiltration/diafiltration procedure and size exclusion chromatography, resulting in highly pure (>95%) PfAMA1-DiCo1, PfAMA1 DiCo2 and PfAMA1 DiCo3, with final yields of 1.8, 1.9 and 1.3 gram, respectively. N-terminal determinations showed that approximately 50% of each of the proteins lost 12 residues from their N-terminus, in accordance with SDS-PAGE (2 main bands) and MS-data. Under reducing conditions a site of limited proteolytic cleavage within a disulphide bonded region became evident. The three proteins quantitatively bound to the mAb 4G2 that recognizes a conformational epitope, suggesting proper folding of the proteins. The lyophilized Drug Product (1:1:1 mixture of PfAMA1-DiCo1, DiCo2, DiCo3) fulfilled all pre-set release criteria (appearance, dissolution rate, identity, purity, protein content, moisture content, sub-visible particles, immuno-potency (after reconstitution with adjuvant), abnormal toxicity, sterility and endotoxin), was stable in accelerated and real-time stability studies at -20°C for over 24 months. When formulated with adjuvants selected for clinical phase I evaluation, the Drug Product did not show adverse effect in a repeated-dose toxicity study in rabbits. The Drug Product has entered a phase Ia/Ib clinical trial.

Characterization of the inhibitory effect of an extract of Prunella vulgaris on Ebola virus glycoprotein (GP)-mediated virus entry and infection

Currently, no approved antiviral therapeutic is available for treatment or prevention of Ebola virus (EBOV) infection. In this study, we characterized an EBOV-glycoprotein (GP) pseudotyped HIV-1-based vector system in different cell cultures, including human umbilical vein endothelial cells (HUVECs) and human macrophages, for the screening of anti-EBOV-GP agent(s). Based on this system, we demonstrated that an aqueous extract (CHPV) from the Chinese herb Prunella vulgaris displayed a potent inhibitory effect on EBOV-GP pseudotyped virus (EBOV-GP-V)-mediated infection in various cell lines, including HUVEC and macrophage. In addition, our results indicated that CHPV was able to block an eGFP-expressing Zaire ebola virus (eGFP-ZEBOV) infection in VeroE6 cells. The anti-EBOV activity of CHPV was exhibited in a dose-dependent manner. At a 12.5 μg/ml concentration, the CHPV showed a greater than 80% inhibition of EBOV-GP-V and eGFP-EBOV infections. Likewise, our studies suggested that the inhibitory effect of CHPV occurred by binding directly to EBOV-GP-Vs and blocking the early viral events. Interestingly, our results have shown that CHPV was able to enhance the anti-EBOV activity of the monoclonal antibody MAb 2G4 against EBOV-GP. Overall, this study provides evidence that CHPV has anti-EBOV activity and may be developed as a novel antiviral approach against EBOV infection.

Generation and characterization of a protective mouse monoclonal antibody against human enterovirus 71.

Human enterovirus 71 (EV71) infection has emerged as a major threat to children; however, no effective antiviral treatment or vaccine is currently available. Antibody-based treatment shows promises to control this growing public health problem of EV71 infection, and a few potent monoclonal antibodies (mAbs) targeting viral capsid protein have been well described. Here, we generated an EV71-specific mouse mAb 2G8 that conferred full protection against lethal EV71 challenge in a suckling mouse model. 2G8 belonged to IgM isotype and neutralized EV71 at the attachment stage. Biochemical assays mapped the binding epitope of 2G8 to the SP70 peptide, which spanning amino acid residues 208-222 on the VP1 protein. Alanine scanning mutagenesis defined the essential roles of multiple residues, including Y208, T210, G212, K215, K218, L220, E221, and Y222, for 2G8 binding. Then, a panel of single mutation was individually introduced into the EV71 infectious clone by reverse genetics, and three mutant viruses, K215A, K218A, and L220A, were successfully recovered and characterized. Biochemical and neutralization assays revealed that K218A mutant partially escaped 2G8 neutralization, while L220A completely abolished 2G8 binding and neutralization. In particular, neutralization assays with human sera demonstrated that K218A and L220A substitutions are also critical for antibody neutralization in natural infection population. These findings not only generate a protective mAb candidate with therapeutic potential but also provide insights into antibody-mediated EV71 neutralization mechanism.

Epitope mapping of 7S cashew antigen in complex with antibody by solution-phase H/D exchange monitored by FT-ICR mass spectrometry.

The potential epitope of a recombinant food allergen protein, cashew Ana o 1, reactive to monoclonal antibody, mAb 2G4, has been mapped by solution-phase amide backbone H/D exchange (HDX) monitored by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Purified mAb 2G4 was incubated with recombinant Ana o 1 (rAna o 1) to form antigen:monoclonal antibody (Ag:mAb) complexes. Complexed and uncomplexed (free) rAna o 1 were then subjected to HDX-MS analysis. Five regions protected from H/D exchange upon mAb binding are identified as potential conformational epitope-contributing segments.

Formation of infectious dengue virus-antibody immune complex in vivo in marmosets (Callithrix jacchus) after passive transfer of anti-dengue virus monoclonal antibodies and infection with dengue virus.

Infection with a dengue virus (DENV) serotype induces cross-reactive, weakly neutralizing antibodies to different dengue serotypes. It has been postulated that cross-reactive antibodies form a virus-antibody immune complex and enhance DENV infection of Fc gamma receptor (FcγR)-bearing cells. We determined whether infectious DENV-antibody immune complex is formed in vivo in marmosets after passive transfer of DENV-specific monoclonal antibody (mAb) and DENV inoculation and whether infectious DENV-antibody immune complex is detectable using FcγR-expressing cells. Marmosets showed that DENV-antibody immune complex was exclusively infectious to FcγR-expressing cells on days 2, 4, and 7 after passive transfer of each of the mAbs (mAb 4G2 and mAb 6B6C) and DENV inoculation. Although DENV-antibody immune complex was detected, contribution of the passively transferred antibody to overall viremia levels was limited in this study. The results indicate that DENV cross-reactive antibodies form DENV-antibody immune complex in vivo, which is infectious to FcγR-bearing cells but not FcγR-negative cells.

A Cronobacter turicensis O1 antigen-specific monoclonal antibody inhibits bacterial motility and entry into epithelial cells.

Cronobacter turicensis is an opportunistic foodborne pathogen that can cause a rare but sometimes lethal infection in neonates. Little is known about the virulence mechanisms and intracellular lifestyle of this pathogen. In this study, we developed an IgG monoclonal antibody (MAb; MAb 2G4) that specifically recognizes the O1 antigen of C. turicensis cells. The antilipopolysaccharide antibody bound predominantly monovalently to the O antigen and reduced bacterial growth without causing cell agglutination. Furthermore, binding of the antibody to the O1 antigen of C. turicensis cells caused a significant reduction of the membrane potential which is required to energize flagellar rotation, accompanied by a decreased flagellum-based motility. These results indicate that binding of IgG to the O antigen of C. turicensis causes a direct antimicrobial effect. In addition, this feature of the antibody enabled new insight into the pathogenicity of C. turicensis. In a tissue culture infection model, pretreatment of C. turicensis with MAb 2G4 showed no difference in adhesion to human epithelial cells, whereas invasion of bacteria into Caco-2 cells was significantly inhibited.

Topical gel formulation of broadly neutralizing anti-HIV-1 monoclonal antibody VRC01 confers protection against HIV-1 vaginal challenge in a humanized mouse model

The new generation broadly neutralizing antibody VRC01 against HIV-1 shows great potential as a topically administered microbicide to prevent sexual transmission. We evaluated its efficacy in a RAG-hu humanized mouse model of vaginal HIV-1 transmission. Mice were challenged vaginally with R5 tropic HIV-1 BaL an hour after intravaginal application of the VRC01 (1mg/ml concentration) gel. A combination of four first generation bNAbs, namely b12, 2F5, 4E10 and 2G12, was used as a positive efficacy control whereas a non-specific dengue MAb 4G2 was used as negative control. Our results showed that seven out of nine VRC01 antibody administered mice and all of the mice receiving the four bNAb antibody combination were protected against HIV-1 challenge. These findings demonstrate the efficacy of the new bNAb VRC01 as a topical microbicide to protect against HIV-1 vaginal transmission and highlight the use of the RAG-hu mouse model for testing HIV prevention strategies.

Functional Analysis of Two Cavities in Flavivirus NS5 Polymerase

Flavivirus NS5 protein encodes methyltransferase and RNA-dependent RNA polymerase (RdRp) activities. Structural analysis of flavivirus RdRp domains uncovered two conserved cavities (A and B). Both cavities are located in the thumb subdomains and represent potential targets for development of allosteric inhibitors. In this study, we used dengue virus as a model to analyze the function of the two RdRp cavities. Amino acids from both cavities were subjected to mutagenesis analysis in the context of genome-length RNA and recombinant NS5 protein; residues critical for viral replication were subjected to revertant analysis. For cavity A, we found that only one (Lys-756) of the seven selected amino acids is critical for viral replication. Alanine substitution of Lys-756 did not affect the RdRp activity, suggesting that this residue functions through a nonenzymatic mechanism. For cavity B, all four selected amino acids (Leu-328, Lys-330, Trp-859, and Ile-863) are critical for viral replication. Biochemical and revertant analyses showed that three of the four mutated residues (Leu-328, Trp-859, and Ile-863) function at the step of initiation of RNA synthesis, whereas the fourth residue (Lys-330) functions by interacting with the viral NS3 helicase domain. Collectively, our results have provided direct evidence for the hypothesis that cavity B, but not cavity A, from dengue virus NS5 polymerase could be a target for rational drug design.

Plant production of anti‐β‐glucan antibodies for immunotherapy of fungal infections in humans

There is an increasing interest in the development of therapeutic antibodies (Ab) to improve the control of fungal pathogens, but none of these reagents is available for clinical use. We previously described a murine monoclonal antibody (mAb 2G8) targeting β-glucan, a cell wall polysaccharide common to most pathogenic fungi, which conferred significant protection against Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans in animal models. Transfer of this wide-spectrum, antifungal mAb into the clinical setting would allow the control of most frequent fungal infections in many different categories of patients. To this aim, two chimeric mouse-human Ab derivatives from mAb 2G8, in the format of complete IgG or scFv-Fc, were generated, transiently expressed in Nicotiana benthamiana plants and purified from leaves with high yields (approximately 50 mg Ab/kg of plant tissues). Both recombinant Abs fully retained the β-glucan-binding specificity and the antifungal activities of the cognate murine mAb against C. albicans. In fact, they recognized preferentially β1,3-linked glucan molecules present at the fungal cell surface and directly inhibited the growth of C. albicans and its adhesion to human epithelial cells in vitro. In addition, both the IgG and the scFv-Fc promoted C. albicans killing by isolated, human polymorphonuclear neutrophils in ex vivo assays and conferred significant antifungal protection in animal models of systemic or vulvovaginal C. albicans infection. These recombinant Abs represent valuable molecules for developing novel, plant-derived immunotherapeutics against candidiasis and, possibly, other fungal diseases.