Live imaging of SARS-CoV-2 infection in mice reveals that neutralizing antibodies require Fc function for optimal efficacy.

Neutralizing antibodies (NAbs) are effective in treating COVID-19, but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment during prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. Real-time imaging revealed that the virus spread sequentially from the nasal cavity to the lungs in mice and thereafter systemically to various organs including the brain, culminating in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days. In addition to direct neutralization, depletion studies indicated that Fc effector interactions of NAbs with monocytes, neutrophils, and natural killer cells were required to effectively dampen inflammatory responses and limit immunopathology. Our study highlights that both Fab and Fc effector functions of NAbs are essential for optimal in vivo efficacy against SARS-CoV-2.

Broadly neutralizing anti-HIV-1 antibodies require Fc effector functions for in vivo activity.

Broadly neutralizing antibodies (bNAbs) against HIV-1 provide both effective pre-exposure prophylaxis and treatment of HIV-1 infection in murine and nonhuman primate models, suggesting their potential use in humans. Although much is known about the role of variable domains in the neutralization breadth and potency of these bNAbs, the contribution of Fc domains to their activities is, by contrast, poorly characterized. Assessment of the in vivo activity of several bNAbs revealed that FcγR-mediated effector function contributes substantially to their capacity to block viral entry, suppress viremia, and confer therapeutic activity. Enhanced in vivo potency of anti-HIV-1 bNAbs was associated with preferential engagement of activating, but not inhibitory FcγRs, and Fc domain-engineered bNAb variants with selective binding capacity for activating FcγRs displayed augmented protective activity. These findings reveal key roles for Fc effector function in the in vivo activity of anti-HIV-1 bNAbs and provide strategies for generating bNAbs with improved efficacy.

A role for the ITAM signaling module in specifying cytokine-receptor functions.

Diverse cellular responses to external cues are controlled by a small number of signal-transduction pathways, but how the specificity of functional outcomes is achieved remains unclear. Here we describe a mechanism for signal integration based on the functional coupling of two distinct signaling pathways widely used in leukocytes: the ITAM pathway and the Jak-STAT pathway. Through the use of the receptor for interferon-γ (IFN-γR) and the ITAM adaptor Fcγ as an example, we found that IFN-γ modified responses of the phagocytic antibody receptor FcγRI (CD64) to specify cell-autonomous antimicrobial functions. Unexpectedly, we also found that in peritoneal macrophages, IFN-γR itself required tonic signaling from Fcγ through the kinase PI(3)K for the induction of a subset of IFN-γ-specific antimicrobial functions. Our findings may be generalizable to other ITAM and Jak-STAT signaling pathways and may help explain signal integration by those pathways.

A Novel Dual-Fc Bispecific Antibody with Enhanced Fc Effector Function.

Bispecific antibodies (BsAbs) are undergoing continued development for applications in oncology and autoimmune diseases. While increasing activity by having more than one targeting arm, most BsAb engineering employs single Fc engagement as monoclonal antibodies. Here, we designed a novel immunoglobulin gamma-1 (IgG1)-derived dual-Fc BsAb containing two Fc regions and two distinct asymmetric antigen binding arms comprising a Fab arm and another VHH domain. In conjunction with the knob-into-hole technology, dual-Fc BsAbs could be produced with a high yield and good stability. We explore how Fc engineering effects on dual-Fc constructs could boost the desired therapeutic efficacy. This new format enabled simultaneous bispecific binding to corresponding antigens. Furthermore, compared to the one-Fc control molecules, dual-Fc BsAbs were shown to increase the avidity-based binding to FcγRs to result in higher ADCC and ADCP activities by potent avidity via binding to two antigens and Fc receptors. Overall, this novel BsAb format with enhanced effector functionalities provides a new option for antibody-based immunotherapy.

Assessment of IgG-Fc glycosylation from individual RhD-specific B cell clones reveals regulation at clonal rather than clonotypic level.

The type and strength of effector functions mediated by immunoglobulin G (IgG) antibodies rely on the subclass and the composition of the N297 glycan. Glycosylation analysis of both bulk and antigen-specific human IgG has revealed a marked diversity of the glycosylation signatures, including highly dynamic patterns as well as long-term stability of profiles, yet information on how individual B cell clones would contribute to this diversity has hitherto been lacking. Here, we assessed whether clonally related B cells share N297 glycosylation patterns of their secreted IgG. We differentiated single antigen-specific peripheral IgG+ memory B cells into antibody-secreting cells and analysed Fc glycosylation of secreted IgG. Furthermore, we sequenced the variable region of their heavy chain, which allowed the grouping of the clones into clonotypes. We found highly diverse glycosylation patterns of culture-derived IgG, which, to some degree, mimicked the glycosylation of plasma IgG. Each B cell clone secreted IgG with a mixture of different Fc glycosylation patterns. The majority of clones produced fully fucosylated IgG. B cells producing afucosylated IgG were scattered across different clonotypes. In contrast, the remaining glycosylation traits were, in general, more uniform. These results indicate IgG-Fc fucosylation to be regulated at the single-clone level, whereas the regulation of other glycosylation traits most likely occurs at a clonotypic or systemic level. The discrepancies between plasma IgG and culture-derived IgG, could be caused by the origin of the B cells analysed, clonal dominance or factors from the culture system, which need to be addressed in future studies.

Chimerism of avian IgY-scFv and truncated IgG-Fc: A novel strategy in cross-species antibody generation and enhancement.

Chicken single-chain fragment variable (IgY-scFv) is a functional fragment and an emerging development in genetically engineered antibodies with a wide range of biomedical applications. However, scFvs have considerably shorter serum half-life due to the absence of antibody Fc region compared with the full-length antibody, and usually requires continuous intravenous administration for efficacy. A promising approach to overcome this limitation is to fuse scFv with immunoglobulin G (IgG) Fc region, for better recognition and mediation by the neonatal Fc receptor (FcRn) in the host. In this study, engineered mammalian ΔFc domains (CH2, CH3, and intact Fc region) were fused with anti-canine parvovirus-like particles avian IgY-scFv to produce chimeric antibodies and expressed in the HEK293 cell expression system. The obtained scFv-CH2, scFv-CH3, and scFv-Fc can bind with antigen specifically and dose-dependently. Surface plasmon resonance investigation confirmed that scFv-CH2, scFv-CH3, and scFv-Fc had different degrees of binding to FcRn, with scFv-Fc showing the highest affinity. scFv-Fc had a significantly longer half-life in mice compared with the unfused scFv. The identified ΔFcs are promising for the development of engineered Fc-based therapeutic antibodies and proteins with longer half-lives. The avian IgY-scFv-mammalian IgG Fc region opens up new avenues for antibody engineering, and it is a novel strategy to enhance the rapid development and screening of functional antibodies in veterinary and human medicine.

The Chicken A and E Blood Systems Arise from Genetic Variation in and around the Regulators of Complement Activation Region.

The tightly linked A and E blood alloantigen systems are 2 of 13 blood systems identified in chickens. Reported herein are studies showing that the genes encoding A and E alloantigens map within or near to the chicken regulator of complement activation (RCA) gene cluster, a region syntenic with the human RCA. Genome-wide association studies, sequence analysis, and sequence-derived single-nucleotide polymorphism information for known A and/or E system alleles show that the most likely candidate gene for the A blood system is C4BPM gene (complement component 4 binding protein, membrane). Cosegregation of single-nucleotide polymorphism-defined C4BPM haplotypes and blood system A alleles defined by alloantisera provide a link between chicken blood system A and C4BPM. The best match for the E blood system is the avian equivalent of FCAMR (Fc fragment of IgA and IgM receptor). C4BPM is located within the chicken RCA on chicken microchromosome 26 and is separated from FCAMR by 89 kbp. The genetic variation observed at C4BPM and FCAMR could affect the chicken complement system and differentially guide immune responses to infectious diseases.

Heparin-binding VEGFR1 variants as long-acting VEGF inhibitors for treatment of intraocular neovascular disorders.

Neovascularization is a key feature of ischemic retinal diseases and the wet form of age-related macular degeneration (AMD), all leading causes of severe vision loss. Vascular endothelial growth factor (VEGF) inhibitors have transformed the treatment of these disorders. Millions of patients have been treated with these drugs worldwide. However, in real-life clinical settings, many patients do not experience the same degree of benefit observed in clinical trials, in part because they receive fewer anti-VEGF injections. Therefore, there is an urgent need to discover and identify novel long-acting VEGF inhibitors. We hypothesized that binding to heparan-sulfate proteoglycans (HSPG) in the vitreous, and possibly other ocular structures, may be a strategy to promote intraocular retention, ultimately leading to a reduced burden of intravitreal injections. We designed a series of VEGF receptor 1 variants and identified some with strong heparin-binding characteristics and ability to bind to vitreous matrix. Our data indicate that some of our variants have longer duration and greater efficacy in animal models of intraocular neovascularization than current standard of care. Our study represents a systematic attempt to exploit the functional diversity associated with heparin affinity of a VEGF receptor.

Improving Antibody Therapeutics by Manipulating the Fc Domain: Immunological and Structural Considerations.

Interactions between the crystallizable fragment (Fc) domain of antibodies and a plethora of cellular Fc receptors (FcRs) or soluble proteins form a critical link between humoral and innate immunity. In particular, the immunoglobulin G Fc domain is critical for the clearance of target cells by processes that include (a) cytotoxicity, phagocytosis, or complement lysis; (b) modulation of inflammation; (c) antigen presentation; (d) antibody-mediated receptor clustering; and (e) cytokine release. More than 30 Fc-engineered antibodies aimed primarily at tailoring these effects for optimal therapeutic outcomes are in clinical evaluation or have already been approved. Nonetheless, our understanding of how FcR engagement impacts various immune cell phenotypes is still largely incomplete. Recent insights into FcR biology coupled with advances in Fc:FcR structural analysis, Fc engineering, and mouse models that recapitulate human biology are helping to fill in existing knowledge gaps. These advances will provide a blueprint on how to fine-tune the Fc domain to achieve optimal therapeutic efficacy.

FcRn-Targeted Mucosal Vaccination against Influenza Virus Infection.

The respiratory tract is constantly exposed to various airborne pathogens. Most vaccines against respiratory infections are designed for the parenteral routes of administration; consequently, they provide relatively minimal protection in the respiratory tract. A vaccination strategy that aims to induce the protective mucosal immune responses in the airway is urgently needed. The FcRn mediates IgG Ab transport across the epithelial cells lining the respiratory tract. By mimicking this natural IgG transfer, we tested whether FcRn delivers vaccine Ags to induce a protective immunity to respiratory infections. In this study, we designed a monomeric IgG Fc fused to influenza virus hemagglutinin (HA) Ag with a trimerization domain. The soluble trimeric HA-Fc were characterized by their binding with conformation-dependent HA Abs or FcRn. In wild-type, but not FcRn knockout, mice, intranasal immunization with HA-Fc plus CpG adjuvant conferred significant protection against lethal intranasal challenge with influenza A/PR/8/34 virus. Further, mice immunized with a mutant HA-Fc lacking FcRn binding sites or HA alone succumbed to lethal infection. Protection was attributed to high levels of neutralizing Abs, robust and long-lasting B and T cell responses, the presence of lung-resident memory T cells and bone marrow plasma cells, and a remarkable reduction of virus-induced lung inflammation. Our results demonstrate for the first time, to our knowledge, that FcRn can effectively deliver a trimeric viral vaccine Ag in the respiratory tract and elicit potent protection against respiratory infection. This study further supports a view that FcRn-mediated mucosal immunization is a platform for vaccine delivery against common respiratory pathogens.

FGF23 contains two distinct high-affinity binding sites enabling bivalent interactions with α-Klotho.

The three members of the endocrine-fibroblast growth factor (FGF) family, FGF19, 21, and 23 are circulating hormones that regulate critical metabolic processes. FGF23 stimulates the assembly of a signaling complex composed of α-Klotho (KLA) and FGF receptor (FGFR) resulting in kinase activation, regulation of phosphate homeostasis, and vitamin D levels. Here we report that the C-terminal tail of FGF23, a region responsible for KLA binding, contains two tandem repeats, repeat 1 (R1) and repeat 2 (R2) that function as two distinct ligands for KLA. FGF23 variants with a single KLA binding site, FGF23-R1, FGF23-R2, or FGF23-wild type (WT) with both R1 and R2, bind to KLA with similar binding affinity and stimulate FGFR1 activation and MAPK response. R2 is flanked by two cysteines that form a disulfide bridge in FGF23-WT; disulfide bridge formation in FGF23-WT is dispensable for KLA binding and for cell signaling via FGFRs. We show that FGF23-WT stimulates dimerization and activation of a chimeric receptor molecule composed of the extracellular domain of KLA fused to the cytoplasmic domain of FGFR and employ total internal reflection fluorescence microscopy to visualize individual KLA molecules on the cell surface. These experiments demonstrate that FGF23-WT can act as a bivalent ligand of KLA in the cell membrane. Finally, an engineered Fc-R2 protein acts as an FGF23 antagonist offering new pharmacological intervention for treating diseases caused by excessive FGF23 abundance or activity.

A combination of two human monoclonal antibodies limits fetal damage by Zika virus in macaques.

Human infection by Zika virus (ZIKV) during pregnancy can lead to vertical transmission and fetal aberrations, including microcephaly. Prophylactic administration of antibodies can diminish or prevent ZIKV infection in animal models, but whether passive immunization can protect nonhuman primates and their fetuses during pregnancy has not been determined. Z004 and Z021 are neutralizing monoclonal antibodies to domain III of the envelope (EDIII) of ZIKV. Together the two antibodies protect nonpregnant macaques against infection even after Fc modifications to prevent antibody-dependent enhancement (ADE) in vitro and extend their half-lives. Here we report on prophylactic coadministration of the Fc-modified antibodies to pregnant rhesus macaques challenged three times with ZIKV during first and second trimester. The two antibodies did not entirely eliminate maternal viremia but limited vertical transmission, protecting the fetus from neurologic damage. Thus, maternal passive immunization with two antibodies to EDIII can shield primate fetuses from the harmful effects of ZIKV.

Comparative Molecular Characterization and Pharmacokinetics of IgG1-Fc and Engineered Fc Human Antibody Variants to Insulin-like Growth Factor 2 Receptor (IGF2R).

Novel therapeutic approaches are much needed for the treatment of osteosarcoma. Targeted radionuclide therapy (TRT) and radioimmunotherapy (RIT) are promising approaches that deliver therapeutic radiation precisely to the tumor site. We have previously developed a fully human antibody, named IF3, that binds to insulin-like growth factor 2 receptor (IGF2R). IF3 was used in TRT to effectively inhibit tumor growth in osteosarcoma preclinical models. However, IF3's relatively short half-life in mice raised the need for improvement. We generated an Fc-engineered version of IF3, termed IF3δ, with amino acid substitutions known to enhance antibody half-life in human serum. In this study, we confirmed the specific binding of IF3δ to IGF2R with nanomolar affinity, similar to wild-type IF3. Additionally, IF3δ demonstrated binding to human and mouse neonatal Fc receptors (FcRn), indicating the potential for FcRn-mediated endocytosis and recycling. Biodistribution studies in mice showed a higher accumulation of IF3δ in the spleen and bone than wild-type IF3, likely attributed to abnormal spleen expression of IGF2R in mice. Therefore, the pharmacokinetics data from mouse xenograft models may not precisely reflect their behavior in canine and human patients. However, the findings suggest both IF3 and IF3δ as promising options for the RIT of osteosarcoma.

Fc γ receptor compositional heterogeneity: Considerations for immunotherapy development.

The antibody-binding crystallizable fragment (Fc) γ receptors (FcγRs) are expressed by leukocytes and activate or suppress a cellular response once engaged with an antibody-coated target. Therapeutic mAbs that require FcγR binding for therapeutic efficacy are now frontline treatments for multiple diseases. However, substantially fewer development efforts are focused on the FcγRs, despite accounting for half of the antibody-receptor complex. The recent success of engineered cell-based immunotherapies now provides a mechanism to introduce modified FcγRs into the clinic. FcγRs are highly heterogeneous because of multiple functionally distinct alleles for many genes, the presence of membrane-tethered and soluble forms, and a high degree of post-translational modification, notably asparagine-linked glycans. One significant factor limiting FcγR improvement is the fundamental lack of knowledge regarding endogenous receptor forms present in the human body. This review describes the composition of FcγRs isolated from primary human leukocytes, summarizes recent efforts to engineer FcγRs, and concludes with a description of potential FcγR features to enrich for enhanced function. Further understanding FcγR biology could accelerate the development of new clinical therapies targeting immune-related disease.

Presence and possible impact of Fcγ receptors on resident kidney cells in health and disease.

Fcγ receptors (FcγRs) bind the Fc fragment of immunoglobulin G (IgG), mostly after IgG opsonizes a bacterial or viral antigen or danger/damage-associated molecule. Consequently, classic FcγRs initiate phagocytosis of the IgG-antigen immune complex and stimulate an immune reaction against the threat. Signals from activating FcγRs (FcγRI, FcγRIIa/c, FcγRIIIa/b) are balanced by inhibitory FcγRIIb and likely also by two FcR-like proteins (FCRL4 and FCRL5). The neonatal Fc receptor (FcRn) recirculates IgG and increases its half-life. The last FcγR that has been identified in humans, tripartite motif-containing protein 21 (TRIM21), acts toward pathogen destruction via the proteasomal or autophagic pathway. The expression of FcγRs occurs almost exclusively in immune cells. However, podocytes, key cells in the glomerular filtration barrier, also possess several features of an immune cell and express receptors for IgG. The presence of FcγRs in glomeruli was analyzed in the Human Protein Atlas project. FcγR occurrence in whole glomeruli or in particular resident kidney cells was also showed in a few original articles. In human podocytes only FcRn has been studied extensively, and the presence and role of other FcγRs remain obscure. Research on the genetic background of kidney diseases revealed a connection between FcγRs and several nephropathies. Investigations of FcγR expression in podocytes appear to be of great clinical importance. The present review discusses the latest literature on FcγRs in kidney cells (especially podocytes), with an emphasis on their involvement in kidney health and disease.

Impact of glycoengineering and antidrug antibodies on the anticancer activity of a plant-made lectin-Fc fusion protein.

Plants are an efficient production platform for manufacturing glycoengineered monoclonal antibodies and antibody-like molecules. Avaren-Fc (AvFc) is a lectin-Fc fusion protein or lectibody produced in Nicotiana benthamiana, which selectively recognizes cancer-associated high-mannose glycans. In this study, we report the generation of a glycovariant of AvFc that is devoid of plant glycans, including the core α1,3-fucose and ß1,2-xylose residues. The successful removal of these glycans was confirmed by glycan analysis using HPLC. This variant, AvFcΔXF , has significantly higher affinity for Fc gamma receptors and induces higher levels of luciferase expression in an antibody-dependent cell-mediated cytotoxicity (ADCC) reporter assay against B16F10 murine melanoma cells without inducing apoptosis or inhibiting proliferation. In the B16F10 flank tumour mouse model, we found that systemic administration of AvFcΔXF , but not an aglycosylated AvFc variant lacking affinity for Fc receptors, significantly delayed the growth of tumours, suggesting that Fc-mediated effector functions were integral. AvFcΔXF treatment also significantly reduced lung metastasis of B16F10 upon intravenous challenge whereas a sugar-binding-deficient mutant failed to show efficacy. Lastly, we determined the impact of antidrug antibodies (ADAs) on drug activity in vivo by pretreating animals with AvFcΔXF before implanting tumours. Despite a significant ADA response induced by the pretreatment, we found that the activity of AvFcΔXF was unaffected by the presence of these antibodies. These results demonstrate that glycoengineering is a powerful strategy to enhance AvFc's antitumor activity.

Manipulation of mRNA translation elongation influences the fragmentation of a biotherapeutic Fc-fusion protein produced in CHO cells.

Mammalian cells, particularly Chinese hamster ovary cells, are the dominant system for the production of protein-based biotherapeutics, however, product degradation, particularly of Fc-fusion proteins, is sometimes observed that impacts the quality of the protein generated. Here, we identify the site of fragmentation of a model immunoglobulin G1 Fc-fusion protein, show that the observed clipping and aggregation are decreased by reduced temperature culturing, that the fragmentation/clipping is intracellular, and that reduced clipping at a lower temperature (<37°C) relates to mesenger RNA (mRNA) translation elongation. We subsequently show that reduced fragmentation can be achieved at 37°C by addition of chemical reagents that slow translation elongation. We then modified mRNA translation elongation speeds by designing different transcript sequences for the Fc-fusion protein based on alternative codon usage and improved the product yield at 37°C, and the ratio of intact to a fragmented product. Our data suggest that rapid elongation results in misfolding that decreases product fidelity, generating a region susceptible to degradation/proteolysis, whilst the slowing of mRNA translation improves the folding, reducing susceptibility to fragmentation. Manipulation of mRNA translation and/or the target Fc-fusion transcript is, therefore, an approach that can be applied to potentially reduce fragmentation of clipping-prone Fc-fusion proteins.

The interplay of protein engineering and glycoengineering to fine-tune antibody glycosylation and its impact on effector functions.

The N-glycan pattern of an IgG antibody, attached at a conserved site within the fragment crystallizable (Fc) region, is a critical antibody quality attribute whose structural variability can also impact antibody function. For tailoring the Fc glycoprofile, glycoengineering in cell lines as well as Fc amino acid mutations have been applied. Multiple glycoengineered Chinese hamster ovary cell lines were generated, including defucosylated (FUT8KO), α-2,6-sialylated (ST6KI), and defucosylated α-2,6-sialylated (FUT8KOST6KI), expressing either a wild-type anti-CD20 IgG (WT) or phenylalanine to alanine (F241A) mutant. Matrix-assisted laser desorption ionization-time of flight mass spectrometry characterization of antibody N-glycans revealed that the F241A mutation significantly increased galactosylation and sialylation content and glycan branching. Furthermore, overexpression of recombinant human α-2,6-sialyltransferase resulted in a predominance of α-2,6-sialylation rather than α-2,3-sialylation for both WT and heavily sialylated F241A antibody N-glycans. Interestingly, knocking out α-1,6-fucosyltransferase (FUT8KO), which removed core fucose, lowered the content of N-glycans with terminal Gal and increased levels of terminal GlcNAc and Man5 groups on WT antibody. Further complement-dependent cytotoxicity (CDC) analysis revealed that, regardless of the production cells, WT antibody samples have higher cytotoxic CDC activity with more exposed Gal residues compared to their individual F241A mutants. However, the FUT8KO WT antibody, with a large fraction of bi-GlcNAc structures (G0), displayed the lowest CDC activity of all WT antibody samples. Furthermore, for the F241A mutants, a higher CDC activity was observed for α-2,6- compared to α-2,3-sialylation. Antibody-dependent cellular cytotoxicity (ADCC) analysis revealed that the defucosylated WT and F241A mutants showed enhanced in vitro ADCC performance compared to their fucosylated counterparts, with the defucosylated WT antibodies displaying the highest overall ADCC activity, regardless of sialic acid substitution. Moreover, the FcγRIIIA receptor binding by antibodies did not always correspond directly with ADCC result. This study demonstrates that glycoengineering and protein engineering can both promote and inhibit antibody effector functions and represent practical approaches for varying glycan composition and functionalities during antibody development.

Preparation and characterization of a high-affinity monoclonal antibody against nerve growth factor.

Nerve growth factor (NGF) is produced and released in injured tissues or chronic pain tissues caused by other diseases. Studies have shown that monoclonal antibodies targeting NGF have a good efficacy in the treatment of osteoarthritis (OA), low back pain and chronic pain, which may be a promising therapy. In this study, DNA sequences of NGF-his and NGF-hFc were synthesized using eukaryotic expression system and subcloned into pTT5 expression vector. After that, NGF proteins were expressed by transient expression in HEK293E cells. We immunized mice with NGF-hFc protein and fused mouse spleen cells to prepare hybridomas. NGF-His protein was used to screen out the hybridoma supernatant that could directly bind to NGF. Antibodies were purified from hybridioma supernatant. Futhermore, via surface plasmon resonance (SPR) screening, six anti-NGF mAbs were screened to block the binding of NGF and TrkA receptor in the treatment of chronic pain. Among them, 58F10G10H showed high affinity (KD = 1.03 × 10-9 M) and even better than that of positive control antibody Tanezumab (KD = 1.53 × 10-9 M). Moreover, the specific reactivity of 58F10G10H was demonstrated by TF-1 cell proliferation activity experiments, competitive binding Enzyme-linked immunosorbent assay (ELISA) and the arthritis animal models in mice, respectively. In conclusion, in this study, a method for the preparation of high-yield NGF-HFC and NGF-His proteins was designed, and a high-affinity monoclonal antibody against NGF with potential for basic research and clinical application was prepared.

A single injection of anti-HIV-1 antibodies protects against repeated SHIV challenges.

Despite the success of potent anti-retroviral drugs in controlling human immunodeficiency virus type 1 (HIV-1) infection, little progress has been made in generating an effective HIV-1 vaccine. Although passive transfer of anti-HIV-1 broadly neutralizing antibodies can protect mice or macaques against a single high-dose challenge with HIV or simian/human (SIV/HIV) chimaeric viruses (SHIVs) respectively, the long-term efficacy of a passive antibody transfer approach for HIV-1 has not been examined. Here we show, on the basis of the relatively long-term protection conferred by hepatitis A immune globulin, the efficacy of a single injection (20 mg kg(-1)) of four anti-HIV-1-neutralizing monoclonal antibodies (VRC01, VRC01-LS, 3BNC117, and 10-1074 (refs 9 - 12)) in blocking repeated weekly low-dose virus challenges of the clade B SHIVAD8. Compared with control animals, which required two to six challenges (median = 3) for infection, a single broadly neutralizing antibody infusion prevented virus acquisition for up to 23 weekly challenges. This effect depended on antibody potency and half-life. The highest levels of plasma-neutralizing activity and, correspondingly, the longest protection were found in monkeys administered the more potent antibodies 3BNC117 and 10-1074 (median = 13 and 12.5 weeks, respectively). VRC01, which showed lower plasma-neutralizing activity, protected for a shorter time (median = 8 weeks). The introduction of a mutation that extends antibody half-life into the crystallizable fragment (Fc) domain of VRC01 increased median protection from 8 to 14.5 weeks. If administered to populations at high risk of HIV-1 transmission, such an immunoprophylaxis regimen could have a major impact on virus transmission.