Marked enhancement of the immunogenicity of plant-expressed IgG-Fc fusion proteins by inclusion of cholera toxin non-toxic B subunit within the single polypeptide.

Immunoglobulin G (IgG)-based fusion proteins have been widely exploited as a potential vaccine delivery platform but in the absence of exogenous adjuvants, the lack of robust immunity remains an obstacle. Here, we report on a key modification that overcomes that obstacle. Thus, we constructed an IgG-Fc vaccine platform for dengue, termed D-PCF, which in addition to a dengue antigen incorporates the cholera toxin non-toxic B subunit (CTB) as a molecular adjuvant, with all three proteins expressed as a single polypeptide. Following expression in Nicotiana benthamiana plants, the D-PCF assembled as polymeric structures of similar size to human IgM, a process driven by the pentamerization of CTB. A marked improvement of functional properties in vitro and immunogenicity in vivo over a previous iteration of the Fc-fusion protein without CTB [1] was demonstrated. These include enhanced antigen presenting cell binding, internalization and activation, complement activation, epithelial cell interactions and ganglioside binding, as well as more efficient polymerization within the expression host. Following immunization of mice with D-PCF by a combination of systemic and mucosal (intranasal) routes, we observed robust systemic and mucosal immune responses, as well as systemic T cell responses, significantly higher than those induced by a related Fc-fusion protein but without CTB. The induced antibodies could bind to the domain III of the dengue virus envelope protein from all four dengue serotypes. Finally, we also demonstrated feasibility of aerosolization of D-PCF as a prerequisite for vaccine delivery by the respiratory route.

Design and Characterization of a Multistage Peptide-Based Vaccine Platform to Target Mycobacterium tuberculosis Infection.

The complex immunopathology ofMycobacterium tuberculosis(Mtb) is one of the main challenges in developing a novel vaccine against this pathogen, particularly regarding eliciting protection against both active and latent stages. Multistage vaccines, which contain antigens expressed in both phases, represent a promising strategy for addressing this issue, as testified by the tuberculosis vaccine clinical pipeline. Given this approach, we designed and characterized a multistage peptide-based vaccine platform containing CD4+ and CD8+ T cell epitopes previously validated for inducing a relevant T cell response against Mtb. After preliminary screening, CFP10 (32-39), GlfT2 (4-12), HBHA (185-194), and PPE15 (1-15) were selected as promising candidates, and we proved that the PM1 pool of these peptides triggered a T cell response in Mtb-sensitized human peripheral blood mononuclear cells (PBMCs). Taking advantage of the use of thiol-maleimide chemoselective ligation, we synthesized a multiepitope conjugate (Ac-CGHP). Our results showed a structure-activity relationship between the conjugation and a higher tendency to fold and assume an ordered secondary structure. Moreover, the palmitoylated conjugate (Pal-CGHP) comprising the same peptide antigens was associated with an enhanced cellular uptake in human and murine antigen-presenting cells and a better immunogenicity profile. Immunization study, conducted in BALB/c mice, showed that Pal-CGHP induced a significantly higher T cell proliferation and production of IFNγ and TNFα over PM1 formulated in the Sigma Adjuvant System.

Rational design and expression of a recombinant plant rhabdovirus glycoprotein for production of immunoreactive murine anti-sera.

Lettuce necrotic yellows virus (LNYV) is a plant rhabdovirus which has a type-1 transmembrane glycoprotein. Here, we describe the generation of murine anti-sera to the glycoprotein. Rational design, expression, and purification of recombinant glycoprotein, termed rLGe, was undertaken using SignalP4.1 and camSol servers to predict signal peptide cleavage and protein solubility. In order to successfully obtain expression in mammalian cells, LNYV glycoprotein native signal peptide was substituted with that of Rabies virus glycoprotein. In addition, rather than expression of the entire molecule, rLGe consisted of the LNYV glycoprotein ectodomain fused to two affinity tags to minimize the risk of protein aggregation, while facilitating detection and purification. rLGe was transiently expressed in mammalian cell culture, purified using affinity column chromatography, and used to immunize mice. Harvested anti-sera were immunoreactive and specific to the naturally occurring glycoprotein as confirmed by western blotting of plant leaf tissue infected with LNYV.

Nanoparticle-Fusion Protein Complexes Protect against Mycobacterium tuberculosis Infection.

Tuberculosis (TB) is the leading cause of death from infectious disease, and the current vaccine, Bacillus Calmette-Guerin (BCG), is inadequate. Nanoparticles (NPs) are an emerging vaccine technology, with recent successes in oncology and infectious diseases. NPs have been exploited as antigen delivery systems and also for their adjuvantic properties. However, the mechanisms underlying their immunological activity remain obscure. Here, we developed a novel mucosal TB vaccine (Nano-FP1) based upon yellow carnauba wax NPs (YC-NPs), coated with a fusion protein consisting of three Mycobacterium tuberculosis (Mtb) antigens: Acr, Ag85B, and HBHA. Mucosal immunization of BCG-primed mice with Nano-FP1 significantly enhanced protection in animals challenged with low-dose, aerosolized Mtb. Bacterial control by Nano-FP1 was associated with dramatically enhanced cellular immunity compared to BCG, including superior CD4+ and CD8+ T cell proliferation, tissue-resident memory T cell (Trm) seeding in the lungs, and cytokine polyfunctionality. Alongside these effects, we also observed potent humoral responses, such as the generation of Ag85B-specific serum IgG and respiratory IgA. Finally, we found that YC-NPs were able to activate antigen-presenting cells via an unconventional IRF-3-associated activation signature, without the production of potentially harmful inflammatory mediators, providing a mechanistic framework for vaccine efficacy and future development.

Plant-expressed Fc-fusion protein tetravalent dengue vaccine with inherent adjuvant properties.

Dengue is a major global disease requiring improved treatment and prevention strategies. The recently licensed Sanofi Pasteur Dengvaxia vaccine does not protect children under the age of nine, and additional vaccine strategies are thus needed to halt this expanding global epidemic. Here, we employed a molecular engineering approach and plant expression to produce a humanized and highly immunogenic poly-immunoglobulin G scaffold (PIGS) fused to the consensus dengue envelope protein III domain (cEDIII). The immunogenicity of this IgG Fc receptor-targeted vaccine candidate was demonstrated in transgenic mice expressing human FcγRI/CD64, by induction of neutralizing antibodies and evidence of cell-mediated immunity. Furthermore, these molecules were able to prime immune cells from human adenoid/tonsillar tissue ex vivo as evidenced by antigen-specific CD4+ and CD8+ T-cell proliferation, IFN-γ and antibody production. The purified polymeric fraction of dengue PIGS (D-PIGS) induced stronger immune activation than the monomeric form, suggesting a more efficient interaction with the low-affinity Fcγ receptors on antigen-presenting cells. These results show that the plant-expressed D-PIGS have the potential for translation towards a safe and easily scalable single antigen-based tetravalent dengue vaccine.

A polymeric immunoglobulin-antigen fusion protein strategy for enhancing vaccine immunogenicity.

In this study, a strategy based on polymeric immunoglobulin G scaffolds (PIGS) was used to produce a vaccine candidate for Mycobacterium tuberculosis. A genetic fusion construct comprising genes encoding the mycobacterial Ag85B antigen, an immunoglobulin γ-chain fragment and the tailpiece from immunoglobulin µ chain was engineered. Expression was attempted in Chinese Hamster Ovary (CHO) cells and in Nicotiana benthamiana. The recombinant protein assembled into polymeric structures (TB-PIGS) in N. benthamiana, similar in size to polymeric IgM. These complexes were subsequently shown to bind to the complement protein C1q and FcγRs with increased affinity. Modification of the N-glycans linked to TB-PIGS by removal of xylose and fucose residues that are normally found in plant glycosylated proteins also resulted in increased affinity for low-affinity FcγRs. Immunization studies in mice indicated that TB-PIGS are highly immunogenic with and without adjuvant. However, they did not improve protective efficacy in mice against challenge with M. tuberculosis compared to conventional vaccination with BCG, suggesting that additional or alternative antigens may be needed to protect against this disease. Nevertheless, these results establish a novel platform for producing polymeric antigen-IgG γ-chain molecules with inherent functional characteristics that are desirable in vaccines.

Molecular engineering and plant expression of an immunoglobulin heavy chain scaffold for delivery of a dengue vaccine candidate.

In order to enhance vaccine uptake by the immune cells in vivo, molecular engineering approach was employed to construct a polymeric immunoglobulin G scaffold (PIGS) that incorporates multiple copies of an antigen and targets the Fc gamma receptors on antigen-presenting cells. These self-adjuvanting immunogens were tested in the context of dengue infection, for which there is currently no globally licensed vaccine yet. Thus, the consensus domain III sequence (cEDIII) of dengue glycoprotein E was incorporated into PIGS and expressed in both tobacco plants and Chinese Ovary Hamster cells. Purified mouse and human cEDIII-PIGS were fractionated by HPLC into low and high molecular weight forms, corresponding to monomers, dimers and polymers. cEDIII-PIGS were shown to retain important Fc receptor functions associated with immunoglobulins, including binding to C1q component of the complement and the low affinity Fcγ receptor II, as well as to macrophage cells in vitro. These molecules were shown to be immunogenic in mice, with or without an adjuvant, inducing a high level IgG antibody response which showed a neutralizing potential against the dengue virus serotype 2. The cEDIII-PIGS also induced a significant cellular immune response, IFN-γ production and polyfunctional T cells in both the CD4+ and CD8+ compartments. This proof-of-principle study shows that the potent antibody Fc-mediated cellular functions can be harnessed to improve vaccine design, underscoring the potential of this technology to induce and modulate a broad-ranging immune response.

Murine IL-4Δ2 splice variant down-regulates IL-4 activities independently of IL-4Rα binding and STAT-6 phosphorylation.

IL-4 is a pleiotropic cytokine that is highly Th2 polarizing. The ratio of IL-4 and its splice variant IL-4Δ2 observed in human health and disease suggests a role for both isoforms. In the present study, the biological function of murine IL-4Δ2 and the potential mechanism of action were studied. We report for the first time the generation of a functional, recombinant murine IL-4Δ2 form which is suggestive of its possible biological role in this species. Recombinant murine IL-4Δ2 inhibited IL-4 mediated cellular processes in macrophages and lymphocytes. Specifically, (i) it reversed IL-4 mediated inhibition of IFN-γ induced nitric oxide release by macrophages, (ii) inhibited IL-4 mediated induction of T cell proliferation, and (iii) prevented IL-4 stimulation of IgE synthesis by B cells. However, IL-4Δ2 did not compete with IL-4 for IL-4Rα binding and did not interfere with the downstream STAT-6 phosphorylation in T cells, suggesting an alternative mechanism for its antagonism of specific IL4-driven effects. These findings suggest that the mouse is a suitable experimental model for studies of the biology of IL-4 and its alternative splice variant.

Oral immunisation of mice with transgenic rice calli expressing cholera toxin B subunit fused to consensus dengue cEDIII antigen induces antibodies to all four dengue serotypes.

Dengue virus (DENV) infection is an emerging global health threat. DENV consists of four distinct serotypes, necessitating a tetravalent vaccine. In this study, expression of consensus envelope protein domain III (cEDIII) fused to cholera toxin B subunit (CTB) in transgenic rice calli was improved using the luminal binding protein BiP at the N-terminus and the SEKDEL signal sequences at the C-terminus, targeting the recombinant protein to endoplasmic reticulum (ER). We found that the fusion protein showed higher levels of expression when compared to the fusion proteins using rice amylase 3D (RAmy3D) or CTB native signal sequence only. The CTB-cEDIII fusion protein was evaluated as an oral dengue vaccine candidate in mice. Serotype specific systemic IgG antibodies and specific IgA response in feces were detected and furthermore, T cell proliferation and high frequency antibody-secreting B cells were detected in the spleen. These results suggest the possible use of plant-based dengue tetravalent vaccine targeted to the mucosal immune system for induction of systemic and mucosal immune responses to DENV infection.

Reconciling Estimates of Cell Proliferation from Stable Isotope Labeling Experiments.

Stable isotope labeling is the state of the art technique for in vivo quantification of lymphocyte kinetics in humans. It has been central to a number of seminal studies, particularly in the context of HIV-1 and leukemia. However, there is a significant discrepancy between lymphocyte proliferation rates estimated in different studies. Notably, deuterated (2)H2-glucose (D2-glucose) labeling studies consistently yield higher estimates of proliferation than deuterated water (D2O) labeling studies. This hampers our understanding of immune function and undermines our confidence in this important technique. Whether these differences are caused by fundamental biochemical differences between the two compounds and/or by methodological differences in the studies is unknown. D2-glucose and D2O labeling experiments have never been performed by the same group under the same experimental conditions; consequently a direct comparison of these two techniques has not been possible. We sought to address this problem. We performed both in vitro and murine in vivo labeling experiments using identical protocols with both D2-glucose and D2O. This showed that intrinsic differences between the two compounds do not cause differences in the proliferation rate estimates, but that estimates made using D2-glucose in vivo were susceptible to difficulties in normalization due to highly variable blood glucose enrichment. Analysis of three published human studies made using D2-glucose and D2O confirmed this problem, particularly in the case of short term D2-glucose labeling. Correcting for these inaccuracies in normalization decreased proliferation rate estimates made using D2-glucose and slightly increased estimates made using D2O; thus bringing the estimates from the two methods significantly closer and highlighting the importance of reliable normalization when using this technique.

Mucosal delivery of antigen-coated nanoparticles to lungs confers protective immunity against tuberculosis infection in mice.

Mucosal boosting of BCG-immunised individuals with a subunit tuberculosis (TB) vaccine would be highly desirable, considering that the lungs are the principal port of entry for Mycobacterium tuberculosis (MTB) and the site of the primary infection and reactivation. However, the main roadblock for subunit TB vaccine development is the lack of suitable adjuvants that could induce robust local and systemic immune responses. Here, we describe a novel vaccine delivery system that was designed to mimic, in part, the MTB pathogen itself. The surface of yellow carnauba wax nanoparticles was coated with the highly immunogenic Ag85B Ag of MTB and they were directed to the alveolar epithelial surfaces by the incorporation of the heparin-binding hemagglutinin adhesion (HBHA) protein. Our results showed that the i.n. immunisation of BCG-primed BALB/c mice with nanoparticles adsorbed with Ag85B-HBHA (Nano-AH vaccine) induced robust humoral and cellular immune responses and IFN-γ production, and multifunctional CD4⁺ T cells expressing IFN-γ, IL-2 and TNF-α. Mice challenged with H37Rv MTB had a significantly reduced bacterial load in their lungs when compared with controls immunised with BCG alone. We therefore conclude that this immunisation approach is an effective means of boosting the BCG-induced anti-TB immunity.

Plant-derived recombinant immune complexes as self-adjuvanting TB immunogens for mucosal boosting of BCG.

Progress with protein-based tuberculosis (TB) vaccines has been limited by poor availability of adjuvants suitable for human application. Here, we developed and tested a novel approach to molecular engineering of adjuvanticity that circumvents the need for exogenous adjuvants. Thus, we generated and expressed in transgenic tobacco plants the recombinant immune complexes (RICs) incorporating the early secreted Ag85B and the latency-associated Acr antigen of Mycobacterium tuberculosis, genetically fused as a single polypeptide to the heavy chain of a monoclonal antibody to Acr. The RICs were formed by virtue of the antibody binding to Acr from adjacent molecules, thus allowing self-polymerization of the complexes. TB-RICs were purified from the plant extracts and shown to be biologically active by demonstrating that they could bind to C1q component of the complement and also to the surface of antigen-presenting cells. Mice immunized with BCG and then boosted with two intranasal immunizations with TB-RICs developed antigen-specific serum IgG antibody responses with mean end-point titres of 1 : 8100 (Acr) and 1 : 24 300 (Ag85B) and their splenocytes responded to in vitro stimulation by producing interferon gamma. 25% of CD4+ proliferating cells simultaneously produced IFN-γ, IL-2 and TNF-α, a phenotype that has been linked with protective immune responses in TB. Importantly, mucosal boosting of BCG-immunized mice with TB-RICs led to a reduced M. tuberculosis infection in their lungs from log10 mean = 5.69 ± 0.1 to 5.04 ± 0.2, which was statistically significant. We therefore propose that the plant-expressed TB-RICs represent a novel molecular platform for developing self-adjuvanting mucosal vaccines.

Demonstrating the utility of the ex vivo murine mycobacterial growth inhibition assay (MGIA) for high-throughput screening of tuberculosis vaccine candidates against multiple Mycobacterium tuberculosis complex strains.

Human tuberculosis (TB) is caused by various members of the Mycobacterium tuberculosis (Mtb) complex. Differences in host response to infection have been reported, illustrative of a need to evaluate efficacy of novel vaccine candidates against multiple strains in preclinical studies. We previously showed that the murine lung and spleen direct mycobacterial growth inhibition assay (MGIA) can be used to assess control of ex vivo mycobacterial growth by host cells. The number of mice required for the assay is significantly lower than in vivo studies, facilitating testing of multiple strains and/or the incorporation of other cellular analyses. Here, we provide proof-of-concept that the murine MGIA can be applied to evaluate vaccine-induced protection against multiple Mtb clinical isolates. Using an ancient and modern strain of the Mtb complex, we demonstrate that ex vivo bacillus Calmette-Guérin (BCG)-mediated mycobacterial growth inhibition recapitulates protection observed in the lung and spleen following in vivo infection of mice. Further, we provide the first report of cellular and transcriptional correlates of BCG-induced growth inhibition in the lung MGIA. The ex vivo MGIA represents a promising platform to gain early insight into vaccine performance against a collection of Mtb strains and improve preclinical evaluation of TB vaccine candidates.

Recombinant immune complexes as vaccines against infectious diseases.

New vaccine technologies are needed to combat many existing infections and prepare better for those that may emerge in the future. The conventional technologies that rely on protein-based vaccines are still severely restricted by the sparsity and poor accessibility of available adjuvants. One possible solution to this problem is to enhance antigen immunogenicity by a more natural means by complexing it with antibodies in the form of immune complexes (ICs). However, natural ICs are impractical as vaccines, and significant research efforts have been made to generate them in recombinant form, with plant bioengineering being at the forefront of these efforts. Here, we describe the challenges and progress made to date to make recombinant IC vaccines applicable to humans.

A modified mycobacterial growth inhibition assay for the functional assessment of vaccine-mediated immunity.

The Mycobacterial growth inhibition assay (MGIA) is an ex-vivo assay used to measure the overall functional immune response elicited by infection or vaccination. In tuberculosis (TB) vaccine development, MGIA is a potentially important tool for preclinical evaluation of early-stage vaccine candidates to complement existing assays, and to potentially reduce the need for lengthy and costly pathogenic Mycobacterium tuberculosis (Mtb) animal challenge experiments. The conventional method of MGIA in mice entails directly infecting mixed cell cultures, most commonly splenocytes, from immunised mice with mycobacteria. However, this direct infection of mixed cell populations may yield unreliable results and lacks sufficient sensitivity to discriminate well between different vaccines due to the low number of mycobacteria-permissive cells. Here, we modified the assay by inclusion of mycobacteria-infected congenic murine macrophage cell lines as the target cells, and by measuring the total number of killed cells rather than the relative reduction between different groups. Thus, using splenocytes from Mycobacterium bovis BCG immunised mice, and J774 and MH-S (BALB/c background) or BL/6-M (C57Bl/6 background) macrophage cell lines, we demonstrated that the modified assay resulted in at least 26-fold greater mycobacterial killing per set quantity of splenocytes as compared to the conventional method. This increased sensitivity of measuring mycobacterial killing was confirmed using both the standard culture forming unit (CFU) assay and luminescence readings of luciferase-tagged virulent and avirulent mycobacteria. We propose that the modified MGIA can be used as a highly calibrated tool for quantitating the killing capacity of immune cells in preclinical evaluation of vaccine candidates for TB.

Implications of O-glycan modifications in the hinge region of a plant-produced SARS-CoV-2-IgA antibody on functionality.

Introduction: Prolyl-4-hydroxylases (P4H) catalyse the irreversible conversion of proline to hydroxyproline, constituting a common posttranslational modification of proteins found in humans, plants, and microbes. Hydroxyproline residues can be further modified in plants to yield glycoproteins containing characteristic O-glycans. It is currently unknown how these plant endogenous modifications impact protein functionality and they cause considerable concerns for the recombinant production of therapeutic proteins in plants. In this study, we carried out host engineering to generate a therapeutic glycoprotein largely devoid of plant-endogenous O-glycans for functional characterization. Methods: Genome editing was used to inactivate two genes coding for enzymes of the P4H10 subfamily in the widely used expression host Nicotiana benthamiana. Using glycoengineering in plants and expression in human HEK293 cells we generated four variants of a potent, SARS-CoV-2 neutralizing antibody, COVA2-15 IgA1. The variants that differed in the number of modified proline residues and O-glycan compositions of their hinge region were assessed regarding their physicochemical properties and functionality. Results: We found that plant endogenous O-glycan formation was strongly reduced on IgA1 when transiently expressed in the P4H10 double mutant N. benthamiana plant line. The IgA1 glycoforms displayed differences in proteolytic stability and minor differences in receptor binding thus highlighting the importance of O-glycosylation in the hinge region of human IgA1. Discussion: This work reports the successful protein O-glycan engineering of an important plant host for recombinant protein expression. While the complete removal of endogenous hydroxyproline residues from the hinge region of plant-produced IgA1 is yet to be achieved, our engineered line is suitable for structure-function studies of O-glycosylated recombinant glycoproteins produced in plants.

Mucosal vaccination against tuberculosis using inert bioparticles.

Needle-free, mucosal immunization is a highly desirable strategy for vaccination against many pathogens, especially those entering through the respiratory mucosa, such as Mycobacterium tuberculosis. Unfortunately, mucosal vaccination against tuberculosis (TB) is impeded by a lack of suitable adjuvants and/or delivery platforms that could induce a protective immune response in humans. Here, we report on a novel biotechnological approach for mucosal vaccination against TB that overcomes some of the current limitations. This is achieved by coating protective TB antigens onto the surface of inert bacterial spores, which are then delivered to the respiratory tract. Our data showed that mice immunized nasally with coated spores developed humoral and cellular immune responses and multifunctional T cells and, most importantly, presented significantly reduced bacterial loads in their lungs and spleens following pathogenic challenge. We conclude that this new vaccine delivery platform merits further development as a mucosal vaccine for TB and possibly also other respiratory pathogens.

A novel human IgA monoclonal antibody protects against tuberculosis.

Abs have been shown to be protective in passive immunotherapy of tuberculous infection using mouse experimental models. In this study, we report on the properties of a novel human IgA1, constructed using a single-chain variable fragment clone (2E9), selected from an Ab phage library. The purified Ab monomer revealed high binding affinities for the mycobacterial α-crystallin Ag and for the human FcαRI (CD89) IgA receptor. Intranasal inoculations with 2E9IgA1 and recombinant mouse IFN-γ significantly inhibited pulmonary H37Rv infection in mice transgenic for human CD89 but not in CD89-negative littermate controls, suggesting that binding to CD89 was necessary for the IgA-imparted passive protection. 2E9IgA1 added to human whole-blood or monocyte cultures inhibited luciferase-tagged H37Rv infection although not for all tested blood donors. Inhibition by 2E9IgA1 was synergistic with human rIFN-γ in cultures of purified human monocytes but not in whole-blood cultures. The demonstration of the mandatory role of FcαRI (CD89) for human IgA-mediated protection is important for understanding of the mechanisms involved and also for translation of this approach toward development of passive immunotherapy of tuberculosis.

Role of phagocyte extracellular traps during Mycobacterium tuberculosis infections and tuberculosis disease processes.

Mycobacterium tuberculosis (M.tb) infections remain one of the most significant causes of mortality worldwide. The current situation shows an emergence of new antibiotic-resistant strains making it difficult to control the tuberculosis (TB) disease. A large part of its success as a pathogen is due to its ability to persist for years or even decades without causing evident clinical manifestations. M.tb is highly successful in evading the host-defense by manipulating host-signalling pathways. Although macrophages are generally viewed as the key cell type involved in harboring M.tb, growing evidence shows that neutrophils also play a fundamental role. Both cells are known to act in multiple ways when encountering an invading pathogen, including phagocytosis, release of cytokines and chemokines, and oxidative burst. In addition, the formation of neutrophil extracellular traps (NETs) and macrophage extracellular traps (METs) has been described to contribute to M.tb infections. NETs/METs are extracellular DNA fibers with associated granule components, which are released upon activation of the cells by the pathogen or by pro-inflammatory mediators. On one hand, they can lead to a protective immune response by entrapment and killing of pathogens. However, on the other hand, they can also play a severe pathological role by inducing tissue damage. Extracellular traps (ETs) produced in the pulmonary alveoli can expand easily and expose tissue-damaging factors with detrimental effects. Since host-directed therapies offer a complementary strategy in TB, the knowledge of NET/MET formation is important for understanding potential protective versus detrimental pathways during innate immune signaling. In this review, we summarize the progress made in understanding the role of NETs/METs in the pathogenesis of TB.

Improving the efficacy of plant-made anti-HIV monoclonal antibodies for clinical use.

Introduction: Broadly neutralising antibodies are promising candidates for preventing and treating Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS), as an alternative to or in combination with antiretroviral therapy (ART). These mAbs bind to sites on the virus essential for virus attachment and entry, thereby inhibiting entry into the host cell. However, the cost and availability of monoclonal antibodies, especially combinations of antibodies, hampers implementation of anti-HIV bNAb therapies in low- to middle- income countries (LMICs) where HIV-1 prevalence is highest. Methods: We have produced three HIV broadly neutralizing antibodies (bNAbs), 10-1074, VRC01 and 3BNC117 in the Nicotiana benthamiana transient expression system. The impact of specific modifications to enhance potency and efficacy were assessed. To prolong half-life and increase bioavailability, a M252Y/S254T/T256E (YTE) or M428L/N434S (LS) mutation was introduced. To increase antibody dependent cellular cytotoxicity (ADCC), we expressed an afucosylated version of each antibody using a glycoengineered plant line. Results: The majority of bNAbs and their variants could be expressed at yields of up to 47 mg/kg. Neither the expression system nor the modifications impacted the neutralization potential of the bNAbs. Afucosylated bNAbs exhibit enhanced ability to bind to FcγRIIIa and trigger ADCC, regardless of the presence of Fc amino acid mutations. Lastly, we demonstrated that Fc-modified variants expressed in plants show enhanced binding to FcRn, which results in a favourable in vivo pharmacokinetic profile compared to their unmodified counterparts. Conclusion: Tobacco plants are suitable expression hosts for anti-HIV bNAbs with increased efficacy and an improved pharmacokinetic profile.