Recombinant Immune Complexes Research Articles

Recombinant immune complexes (rICs) as a therapeutic for Multiple Sclerosis (MS): effect on myeloid cell activation

Abstract Monocyte recruitment into the central nervous system (CNS) is critical for driving pathology in neuroinflammatory diseases such as MS. The monocytic influx as well as activation of resident myeloid cells contribute to recruitment and subsequent re-activation of autoreactive T cells, ultimately leading to demyelinating lesions, axonal damage, and progressive paralysis. Using the murine model for MS, experimental autoimmune encephalitis (EAE) we tested a rIC, M019, to determine its efficacy in reducing myeloid cell activation and subsequently reducing disease severity. Four days following induction of EAE with MOG peptide, daily treatment with M019 inhibited clinical symptoms including weight loss and demyelination in a dose-dependent manner. M019 also delayed or prevented disease in the SJL model of relapsing remitting EAE demonstrating its effectiveness against several forms of EAE. Analysis of the immune cell infiltrate in the spinal cords of M019 and vehicle treated mice revealed that M019 significantly inhibits the infiltration of myeloid and lymphoid cells into the CNS. The myeloid cells that were present in the CNS of M019 treated mice had reduced levels of MHC II suggesting they are inefficient at antigen presentation to autoreactive T cells. Microglia in the M019 treated EAE mice did not upregulate MHC II expression compared to the vehicle treated mice suggesting M019 inhibits their activation. M019 binds directly to monocytes in the periphery via CD16 (FcγRIII) and CD32 (FcγRII) and we detected increased chemokines in the sera of M019 treated EAE mice compared to vehicle treated EAE mice. These results suggest that M019 blocks immune cell migration into the CNS, potentially via phenotypic and functional changes to monocytes.

Therapeutic recombinant immune complexes (rICs) inhibit T cell recruitment to the spinal cord during Experimental Autoimmune Encephalomyelitis (EAE)

Abstract Multiple sclerosis (MS) is an autoimmune neurodegenerative disease mediated by autoreactive Th1 and Th17 cells that migrate into the central nervous system (CNS) and target myelin proteins ultimately leading to neuronal damage. Current immunomodulatory treatments reduce disease severity but do not cure the disease, and they do not work for all patients. Recombinant ICs recapitulate the anti-inflammatory properties of intravenous immunoglobulin and may be a viable therapy for MS. The objective of our study was to determine if a rIC, M019, could inhibit autoreactive T cell recruitment into the CNS using the EAE model. To demonstrate that M019 could inhibit disease severity when given therapeutically, C57BL/6 mice were immunized with a CFA-MOG 35–55emulsion followed by pertussis toxin to induce EAE. Upon symptom development, mice were randomly assigned to either the vehicle or M019 treatment groups and treated with M019 or vehicle daily. Mice treated with M019 exhibited reduced clinical scores and lethality compared to vehicle treated mice. Flow cytometric analysis of T cell infiltration into the CNS revealed that M019 inhibited CD4 +and CD8 +T cell infiltration compared to vehicle treated mice with EAE. However, spleen cells from M019 treated mice had similar levels of Th17 cells and increased Th1 cells compared to the vehicle treated group. These results suggest that M019 is not inhibiting Th17 responses in the periphery but is inhibiting their recruitment into the CNS. The effect of M019 is likely indirect, as in vivo drug binding studies show that M019 binds strongly to monocytes with low level binding to T cells. Our studies suggest that M019 may be an effective therapeutic for MS patients.

A Highly Expressing, Soluble, and Stable Plant-Made IgG Fusion Vaccine Strategy Enhances Antigen Immunogenicity in Mice Without Adjuvant

Therapeutics based on fusing a protein of interest to the IgG Fc domain have been enormously successful, though fewer studies have investigated the vaccine potential of IgG fusions. In this study, we systematically compared the key properties of seven different plant-made human IgG1 fusion vaccine candidates using Zika virus (ZIKV) envelope domain III (ZE3) as a model antigen. Complement protein C1q binding of the IgG fusions was enhanced by: 1) antigen fusion to the IgG N-terminus; 2) removal of the IgG light chain or Fab regions; 3) addition of hexamer-inducing mutations in the IgG Fc; 4) adding a self-binding epitope tag to create recombinant immune complexes (RIC); or 5) producing IgG fusions in plants that lack plant-specific β1,2-linked xylose and α1,3-linked fucose N-linked glycans. We also characterized the expression, solubility, and stability of the IgG fusions. By optimizing immune complex formation, a potently immunogenic vaccine candidate with improved solubility and high stability was produced at 1.5 mg IgG fusion per g leaf fresh weight. In mice, the IgG fusions elicited high titers of Zika-specific antibodies which neutralized ZIKV using only two doses without adjuvant, reaching up to 150-fold higher antibody titers than ZE3 antigen alone. We anticipate these findings will be broadly applicable to the creation of other vaccines and antibody-based therapeutics.

Vaccine synergy with virus-like particle and immune complex platforms for delivery of human papillomavirus L2 antigen

Diverse HPV subtypes are responsible for considerable disease burden worldwide, necessitating safe, cheap, and effective vaccines. The HPV minor capsid protein L2 is a promising candidate to create broadly protective HPV vaccines, though it is poorly immunogenic by itself. To create highly immunogenic and safe vaccine candidates targeting L2, we employed a plant-based recombinant protein expression system to produce two different vaccine candidates: L2 displayed on the surface of hepatitis B core (HBc) virus-like particles (VLPs) or L2 genetically fused to an immunoglobulin capable of forming recombinant immune complexes (RIC). Both vaccine candidates were potently immunogenic in mice, but were especially so when delivered together, generating very consistent and high antibody titers directed against HPV L2 (>1,000,000) that correlated with virus neutralization. These data indicate a novel immune response synergy upon co-delivery of VLP and RIC platforms, a strategy that can be adapted generally for many different antigens.

Novel vaccination approach for dengue infection based on recombinant immune complex universal platform

Dengue infection is on the rise in many endemic areas of the tropics. Vaccination remains the most realistic strategy for prevention of this potentially fatal viral disease but there is currently no effective vaccine that could protect against all four known serotypes of the dengue virus. This study describes the generation and testing of a novel vaccination approach against dengue based on recombinant immune complexes (RIC). We modelled the dengue RIC on the existing Ebola RIC (Phoolcharoen, et al. Proc Natl Acad Sci USA 2011;108(Dec (51)):20695) but with a key modification that allowed formation of a universal RIC platform that can be easily adapted for use for other pathogens. This was achieved by retaining only the binding epitope of the 6D8 ant-Ebola mAb, which was then fused to the consensus dengue E3 domain (cEDIII), resulting in a hybrid dengue-Ebola RIC (DERIC). We expressed human and mouse versions of these molecules in tobacco plants using a geminivirus-based expression system. Following purification from the plant extracts by protein G affinity chromatography, DERIC bound to C1q component of complement, thus confirming functionality. Importantly, following immunization of mice, DERIC induced a potent, virus-neutralizing anti-cEDIII humoral immune response without exogenous adjuvants. We conclude that these self-adjuvanting immunogens have the potential to be developed as a novel vaccine candidate for dengue infection, and provide the basis for a universal RIC platform for use with other antigens.

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 1:24300 (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.

Highly Immunogenic and Protective Recombinant Vaccine Candidate Expressed in Transgenic Plants

Vaccine development has been hampered by difficulties in developing new and safe adjuvants, so alternative technologies that offer new avenues forward are urgently needed. The goal of this study was to express a monoclonal recombinant immune complex in a transgenic plant. A recombinant protein consisting of a tetanus toxin C fragment-specific monoclonal antibody fused with the tetanus toxin C fragment was designed and expressed. Immune complex formation occurred between individual fusion proteins to form immune complex-like aggregates that bound C1q and FcgammaRIIa receptor and could be targeted to antigen-presenting cells. Unlike antigen alone, the recombinant immune fusion complexes were highly immunogenic in mice and did not require coadministration of an adjuvant (when injected subcutaneously). Indeed, these complexes elicited antibody titers that were more than 10,000 times higher than those observed in animals immunized with the antigen alone. Furthermore, animals immunized with only 1 mug of recombinant immune complex without adjuvant were fully protected against lethal challenge. This the first report on the use of a genetic fusion between antigen and antibody to ensure an optimal expression ratio between the two moieties and to obtain fully functional recombinant immune complexes as a new vaccine model.