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HIV-1 and influenza antigens synthetically linked to IgG2a Fc elicit superior humoral responses compared to unmodified antigens in mice
Abstract
Using murine IgG subclass molecules (IgG1 or IgG2a) synthetically fused to HIV-1 or influenza test antigens, we explored the potential for IgG Fc scaffolds to augment immunogenicity. Each antigen (Ag) was grafted onto a hinge-Fc scaffold containing all critical residues necessary for interaction with effector cells, thus retaining effector functions of the native IgG subclass. We hypothesized that the differential affinity of FcγRs for specific IgG subclasses would influence the magnitude of immune responses elicited by immunization with an Ag-IgG Fc fusion vaccine. We demonstrate here that the antigen-specific humoral response elicited by Ag-IgG2a fusion vaccines is at least tenfold greater than that elicited by native antigen, that this response is superior to that elicited by Ag-IgG1, and that the augmented antigen-specific humoral response elicited is Fcγ receptor-dependent.
... The Fc receptors for IgG (FccRs), expressed on dendritic cells (DCs) and APCs, can bind and internalize antigen-IgG immune complexes via the interaction with the IgG, resultsing in enrichment of exogenous antigens in DCs, which facilitates DC maturation and antigen-specific T cell responses and humoral responses. Recombinant antigen-immunoglobin Fc-fusion proteins were shown to increase the immunogenicity of the fused antigens and elicit neutralizing antibody responses to HIV [29,30] and protective immunity to virulent herpes simplex virus [31], influenza viruses [32] and Ebola viruses [33]. In this study, we showed for the first time that fusing HPV16 L2 aa 17-36 epitope repeats to a recombinant ligand for FccRs (designated L2R4, see Figure 1A-B) could significantly increase the immunogenicity of the L2 peptide and induce cross-neutralizing antibodies and protective immunity against a range of phylogenetically distant HPV types. ...
... In the current study, we used R4, a modified IgG1 Fc, as a scaffold to display L2 epitopes and observed strong effects of R4 scaffold on enhancing the immune responses induced by L2 epitopes. Fc of IgG is considered as an important fusion tag for coexpressing several viral proteins to promote correct folding of the fusion proteins, facilitate purification, enhance the binding to APCs expressing FccR and improve the immunogenicity of fused proteins [30,32,33,46,47]. Previous studies showed that recombinant proteins by fusing R4 to antigen proteins, such as human serum albumin domain 1 and the clostridal botulinum neurotoxin, could be effectively targeted to all classes of FccRs on APCs and elicit enhanced antigen-reactive antibody responses [34,35]. ...
Current human papillomavirus (HPV) major capsid protein L1 virus-like particles (VLPs)-based vaccines in clinic induce strong HPV type-specific neutralizing antibody responses. To develop pan-HPV vaccines, here, we show that the fusion protein E3R4 consisting of three repeats of HPV16 L2 aa 17-36 epitope (E3) and a modified human IgG1 Fc scaffold (R4) induces cross-neutralizing antibodies and protective immunity against divergent HPV types. E3R4 was expressed as a secreted protein in baculovirus expression system and could be simply purified by one step Protein A affinity chromatography with the purity above 90%. Vaccination of E3R4 formulated with Freunds adjuvant not only induced cross-neutralizing antibodies against HPV pseudovirus types 16, 18, 45, 52, 58, 6, 11 and 5 in mice, but also protected mice against vaginal challenges with HPV pseudovirus types 16, 45, 52, 58, 11 and 5 for at least eleven months after the first immunization. Moreover, vaccination of E3R4 formulated with FDA approved adjuvant alum plus monophosphoryl lipid A also induced cross-neutralizing antibodies against HPV types 16, 18 and 6 in rabbits. Thus, our results demonstrate that delivery of L2 antigen as a modified Fc-fusion protein may facilitate pan-HPV vaccine development.
... The second design concept of the bipartite carrier protein is based on the Fc-mediated antigen uptake/presentation for T helper activation. Previous studies have demonstrated that antigens fused with the IgG Fc region increase the Fc receptor-mediated antigen uptake by APCs, resulting in type II T helper activation and subsequent class switching and affinity maturation of B cells [36][37][38]. In addition, fusion of antigen to IgG Fc can additionally extend the serum half-life of the antigen [38,39]. ...
... IgG Fc-antigen fusion) is more specific for antigen presenting cells (APCs) while the latter is not expected to exhibit any specificity [13,40,41]. Consequently, the former is likely to specifically induce antibody response through activation of type II T helper cells and hence the IgG antibody response [13,36,42] while the latter may also elicit cell-mediated immunity leading to perhaps tissue damage [40]. ...
Development of successful vaccines against glycotopes remains a major challenge. In the current studies, we have successfully developed a novel carrier protein for glycotopes based on the concept of antigen clustering and specific stimulation of T helper cells to mount strong antibody response to glycotopes. The bipartite carrier protein consists of a tandem repeat of a cysteine-rich peptide for docking of clustered glycotopes to effectively activate B cells and an Fc domain for antigen delivery to antigen presenting cells (APCs). To demonstrate its utility, we conjugated the tumor-specific monosaccharide antigen Tn to this novel carrier protein and successfully developed a Tn vaccine against cancer in animal models. The Tn vaccine effectively elicited high-titer IgG1 antibodies against Tn in immunized mice, and effectively suppressed the development of prostate cancer in Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice. Our results suggest that this novel bipartite carrier protein could be effectively used for developing anti-glycotope vaccines such as the anticancer Tn vaccine.
... Perhaps the most important, the addition of the Fc domain remarkably increases the half-life of Fc-fusion proteins (FCPs) in serum, which prolongs therapeutic activity, owing to their interaction with the salvage neonatal Fc receptors (FcRs) [6][7][8]. Moreover, the attached Fc domain also affords FCPs to interact with the FcRs expressed on immune cells, a feature that is particularly essential for their use in oncological therapies and vaccines [9][10][11]. The presence of the Fc domain can also improve solubility, stability, avidity, and potency of FCPs. ...
Fc-fusion proteins (FCPs), a new generation biological medicine, have revolutionized the practice of medicines that treat diseases. However, complex manufacturing techniques are required for FCP production, casting the affordability and accessibility issues in low- and middle-income economies (LMIEs). Virus-vectored system may serve as a simple and cost-effective platform for FCP delivery. As a proof-of-concept study, Newcastle disease virus (NDV), a widely-used vector for vaccine generation, was used as a vector to express and deliver a model FCP composed of the hemagglutinin (HA) and IgG Fc. A recombinant NDV expressing the HA-Fc fusion protein was generated using reverse genetics, which had comparable replication and virulence to the parental virus. High levels of expression of soluble HA-Fc were detected in cell culture and embryonated chicken eggs inoculated with the recombinant NDV. In addition, the recombinant NDV replicated in the lung of mouse, delivering the HA-Fc protein to this organ. The HA-Fc expressed by NDV specifically bound to murine FcγRI, which was dependent on the presence of the Fc tag. The recombinant NDV induced high vector-specific antibody response, whereas it failed to elicit H7N9-specific antibody immunity in mice. The absence of HA-specific antibodies may be attributed to deficient incorporation of the HA-Fc protein into NDV virion particles. Our results indicated that NDV may be potentially used as a vector for FCP expression and delivery. This strategy may help to enhance the affordability and equal accessibility of FCP biological medicines, especially in LIMEs.
Graphical Abstract
... B cells and T cell epitopes of human bFGF/VEGFA, and in CD scanning, the epitopes were abundant in alpha helix, anti-parallel, parallel, beta sheet, and random coil. The effector peptides fused with Fc domain are highly immunogenic to induce immune responses, relative to native antigens (32). The Peptibody we designed could elicit high-titer anti-bFGF/VEGFA antibodies, T cells, and Th1/Th2-type cytokine activation in mice, suggesting that it could stimulate specific humoral and cellular immune responses. ...
Tumor angiogenesis is dependent on growth factors, and inhibition of their pathways is one of the promising strategies in cancer therapy. However, resistance to single pathway has been a great concern in clinical trials so that it necessitates multiple targetable factors for developing tumor angiogenesis inhibitors. Moreover, the strategy of Fc fusion protein is an attractive platform for novel peptide agents, which gains increasing importance with FDA approval because of better immunogenicity and stability. Here, we applied the Fc fusion protein concept to bFGF/VEGFA pathways and designed a multi-epitope Peptibody with immunogenic peptides derived from human bFGF and VEGFA sequences. Immunization with Peptibody could elicit high-titer anti-bFGF and anti-VEGFA antibodies, activate T cells, and induce Th1/Th2-type cytokines. In in vitro experiments, the isolated anti-Peptibody antibody inhibited the proliferation and migration of A549 cells and human umbilical vein endothelial cells (HUVECs) by decreasing the MAPK/Akt/mTOR signal pathways. In the murine tumor model, pre-immunization with Peptibody suppressed the tumor growth and neovascularization of lung cancer by decreasing the production of bFGF/VEGFA/PDGF, the MAPK/Akt/mTOR signal pathways, and the activation of suppressive cells in tumor sites. Further, the biological characterizations of the recombinant Peptibody were investigated systematically, including protein primary structure, secondary structure, stability, and toxicity. Collectively, the results highlighted the strategy of bFGF/VEGFA pathways and Fc fusion protein in suppressing tumor progression and angiogenesis, which emphasized the potential of multiple targetable factors for producing enduring clinical responses in tumor patients.
... Then, the stimulation of humoral immune responses was examined in our study. Reportedly, Zaharatos et al. discovered that HIV-1 and influenza antigens synthetically linked to IgG2a Fc could cause potent humoral responses in mice (Zaharatos et al., 2011). Furthermore, Hong et al. proved that lentivector expressing HBsAg and immunoglobulin Fc fusion antigen could induce humoral immune responses (Hong et al., 2011). ...
Molecular adjuvants are vaccine delivery vehicle to increase specific antigens effectiveness. Herein, we concentrated on IgG Fc, an effective molecular adjuvant, to develop novel pseudorabies virus (PRV) subunit vaccines. Two major protective antigen genes of PRV were constructed and linked into the mouse IgG Fc fragment. The gD, gD-IgG2aFc, gB and gB-IgG2aFc proteins were expressed using a baculovirus system. Mice intranasally immunized with gD-IgG2aFc or gB-IgG2aFc subunit vaccine exhibited significantly higher PRV-specific antibodies, neutralizing antibodies and intracellular cytokines than the mice intranasally immunized with gD or gB subunit vaccine. Moreover, no histopathological lesions were observed in mice immunized with gB-IgG2aFc subunit vaccine via histopathology examination. Further, the gB-IgG2aFc subunit vaccine was efficient for PRV infection compared with live attenuated vaccine. Overall, these results suggest that IgG2a Fc fragment, as a potential molecular adjuvant, fused with PRV antigen might be a promising and efficient PRV vaccine candidate.
... We have also previously identified other scaffolds for presenting V1V2 epitopes, including 1FD6-monkey immunoglobulin G (IgG) Fc (NHP group 4; Figure 1C), which was designed to increase the interaction with macaque Fcγ receptors on immune cells. Fusing HIV proteins to IgG Fc has been tested in mice, resulting in improved antibody responses (Chen et al., 2007;Qi et al., 2010;Zaharatos et al., 2011), including increased potency and breadth of neutralizing and ADCC activities in macaques (Shubin et al., 2017). Binding of immune complexes to Fcγ receptors on dendritic cells has also been shown to enhance antigen presentation (Akiyama et al., 2003;Regnault et al., 1999;Sallusto and Lanzavecchia, 1994;Song et al., 1999) and promote somatic hypermutation and affinity maturation (Allen and Cyster, 2008;Aydar et al., 2005;Wu et al., 2008). ...
The V1V2 region of the HIV-1 envelope is the target of several broadly neutralizing antibodies (bNAbs). Antibodies to V1V2 elicited in the RV144 clinical trial correlated with a reduced risk of HIV infection, but these antibodies were without broad neutralizing activity. Antibodies targeting V1V2 also correlated with a reduced viral load in immunized macaques challenged with simian immunodeficiency virus (SIV) or simian/human immunodeficiency virus (SHIV). To focus immune responses on V1V2, we engrafted the native, glycosylated V1V2 domain onto five different multimeric scaffold proteins and conducted comparative immunogenicity studies in macaques. Vaccinated macaques developed high titers of plasma and mucosal antibodies that targeted structurally distinct V1V2 epitopes. Plasma antibodies displayed limited neutralizing activity but were functionally active for ADCC and phagocytosis, which was detectable 1-2 years after immunizations ended. This study demonstrates that multivalent, glycosylated V1V2-scaffold protein immunogens focus the antibody response on V1V2 and are differentially effective at inducing polyfunctional antibodies with characteristics associated with protection.
... Fc fusion proteins can form stable dimers through disulfide bonds in Fc hinges and Fc regions can be folded independently to ensure the stability of chaperone molecules in vivo and in vitro [34]. Fc fragments can also bind to the Fc gamma R (FcγR) on the surface of immune cells and play a variety of biological functions, such as mediating the transport through placental and mucosal barriers, inflammatory response, antibody-dependent cell-mediated phagocytosis (ADCP), antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) [35,36]. In addition, fusion with Fc can improve the expression of protein in mammalian cells and can specifically bind to the Protein A affinity column, simplifying the purification process of Fc fusion protein, which is of great significance in the development of biological products [37,38]. ...
AP25 is an anti-tumor peptide with a high affinity for integrins. It exerts its anti-tumor activity by inhibiting angiogenesis and by directly inhibiting the growth of tumor cells. Its half-life time in vivo is only about 50 minutes, which limits its clinical application. In order to prolong the half-life time of AP25 while preserving its anti-tumor activity, several fusion proteins of AP25 and IgG4 Fc were designed and expressed; their anti-tumor activity and pharmacokinetics properties were evaluated. Firstly, four AP25-Fc fusion protein sequences were designed, and the corresponding proteins were expressed and purified. Based on the results of HUVEC migration inhibition assay, HUVEC and tumor cell proliferation inhibition assay and yields of expression by HEK293 cells, the fusion protein designated PSG4R was selected for further evaluation. The anti-tumor effect of PSG4R was then evaluated in vivo on HCT-116 nude mice xenograft model. And the pharmacokinetics properties of PSG4R were investigated in rats. The results showed that PSG4R could inhibit the growth of xenografts of human colon cancer cell line HCT-116 in nude mice by intravenous administration of 40 mg/kg once every two days. The half-life time of PSG4R was 56.270 ± 15.398 h. This study showed that the construction of AP25-Fc fusion protein could significantly prolong the half-life of AP25 while retaining its anti-tumor activity, which provides a new direction for new drug development of AP25.
... Previous immunization studies in mice tested recombinant proteins where HIV proteins were fused to IgG Fc and showed improved immune responses. When p55 Gag or Env V3 loop was fused to murine Fc from the IgG2a subclass, which binds with the highest affinity to activating Fc gamma receptors (Fc␥Rs) in mice, immune responses were stronger than when Fc from the weakly binding subclass IgG1 Fc was used (5). Mice immunized with gp41 prehairpin fusion intermediate attached to Fc developed neutralizing antibody responses against HIV IIIb (6). ...
A goal for HIV prevention programs is to develop safe and effective vaccines that elicit durable and broadly protective antibodies. Many vaccine programs focus on the immune responses to critical epitopes in the gp120 portion of HIV envelope glycoprotein (Env) and seek to improve the quality and quantity of antibodies by altering the sequence, conformation, oligomerization or glycosylation of gp120 to activate appropriate germline B cells and mimic the subsequent maturation pathways seen in infected individuals. As a complement to these strategies, we developed dimeric fusion protein immunogens consisting of HIVBaL gp120 monomer attached to a Gly/Ser linker that is in turn, fused to one half of the dimeric Fc domain from rhesus macaque IgG1 (Env-rFc). We envisioned that Env-rFc may mimic some aspects of immune complexes by binding Fc gamma receptors (FcγRs) on immune cells to increase the strength, breadth, and durability of Env-specific antibody responses. The Env-rFc retained a capacity to bind both cell surface CD4 and FcγRs. In a rhesus macaque immunization study, Env-rFc elicited higher gp120 binding antibody titers compared to Env and elicited antibodies that recognize CD4-induced epitopes. Env-rFc also induced antibodies capable of neutralizing Tier 1A HIV pseudotyped viruses and mediating antibody-dependent cellular cytotoxicity, outcomes not observed with monomeric gp120 in our study. Serum antibodies produced in Env-rFc immunized macaques had increased durability compared to Env monomer immunization. Our work suggests that adding IgG1 Fc to Env-based immunogens may stimulate increased effector capacity in the immune sera and improve the protective serum antibody response.
... Currently, nine human IgG1 fragment crystallizable (Fc) domain fusion drugs have been approved by the FDA to extend the serum half-lives of the linked antigens (Rath et al., 2015). Moreover, antigens synthetically linked to immunoglobulin IgG Fc molecules are more immunogenic than native antigens alone (Konduru et al., 2011;Zaharatos et al., 2011;Tayra et al., 2013;Iorio et al., 2015). Thus, the introduction of immunoglobulin Fc can definitely improve the immune effect of subunit vaccines. ...
Fc-fusion technologies, in which immunoglobulin Fc is genetically fused to an antigenic protein, have been developed to confer antibody-like properties to proteins and peptides. Mammalian IgG Fc fusion exhibits improved antigen-induced immune responses by providing aggregates with high avidity for the IgG Fc receptor and salvaging the antigenic portion from endosomal degradation. However, whether the linked chicken IgY Fc fragment shares similar characteristics to mammalian IgG Fc remains unclear. In this study, we linked the chicken IgY Fc gene to the outer membrane protein A (ompA) of Bordetella avium through overlapping PCR. The fusion gene was cloned into the pPIC9 plasmid to construct the recombinant Pichia pastoris transformant expressing the ompA–Fc fusion protein. The effects of the linked Fc on macrophage vitality, activity, efficiency of antigen processing, and immune responses induced by the fused ompA were investigated. Furthermore, the effect of Taishan Pinus massoniana pollen polysaccharide (TPPPS), an immunomodulator, on chicken macrophage activation was evaluated. TPPPS was also used as an adjuvant to investigate its immunomodulatory effect on immunoresponses induced by the fused ompA–Fc in chickens. The pinocytosis, phagocytosis, secretion of nitric oxide and TNF-α, and MHC-II molecular expression of the macrophages treated with the fused ompA–Fc were significantly higher than those of the macrophages treated with ompA alone. The addition of TPPPS to the fused ompA–Fc further enhanced macrophage functions. The fused ompA–Fc elicited higher antigen-specific immune responses and protective efficacy compared with ompA alone. Moreover, the fused ompA–Fc conferred higher serum antibody titers, serum IL-2 and IL-4 concentrations, CD4+ and CD8+ T-lymphocyte counts, lymphocyte transformation rate, and protection rate compared with ompA alone. Notably, the prepared TPPPS adjuvant ompA–Fc vaccines induced high immune responses and protection rate. The linked Fc and TPPPS adjuvant can remarkably enhance macrophage functions and specific immune responses. This study provides new perspectives to improve the immune effects of subunit vaccines for prevention of poultry diseases.
This study compares the effect of recombinant Factor VIII Fc fusion protein (rFVIII-Fc) with recombinant FVIII (rFVIII) on monocyte-derived dendritic cells (moDC's). Cells treated with rFVIII-Fc showed morphological changes typical for cell activation, had a significant up-regulation of cell activation markers and produced higher levels of pro-inflammatory cytokines. Even after stimulation with Lipopolysaccharides, the addition of rFVIII-Fc led to increased expression of activation markers, indicating that rFVIII-Fc is capable of amplifying the maturation signal. On the contrary, cultivation of moDC's with rFVIII did not alter cell morphology or increase surface activation marker expression and pro-inflammatory cytokine production. The binding of the Fc domain to the activating Fcγ receptor IIa (FcγRIIa) can cause cell activation. Therefore, the effect of rFVIII-Fc on FcγRIIa was analyzed in detail. Cultivation of moDC's with rFVIII-Fc led to increased phosphorylation of FcγRIIa, which was not detected for rFVIII. Blocking FcγRIIa prior to the cultivation with rFVIII-Fc significantly reduced the activating effect of rFVIII-Fc, indicating that rFVIII-Fc-induced moDC activation was caused by FcγRIIa. Moreover, rFVIII-Fc bound to FCGR2A-transfected human embryonic kidney 293 cells. Taken together, our data present a new mechanism of moDC activation by rFVIII-Fc via FcγRIIa.
Background:
Fc-fusion is a platform technology used to increase the circulating half-life of protein and peptide therapeutics. However, there are potential immunological consequences with this approach, such as changes in the molecule's immunogenicity as well as possible interactions with a repertoire of Fc-receptors (FcR) which can modulate immune responses.
Objectives/methods:
Using a mouse hemophilia B (HB) model, we compared the immune responses to infusions of recombinant human Factor IX (hFIX) and hFIX fused to mouse IgG2a-Fc (hFIX-mFc). The mFc was employed to allow species-specific Fc-FcγR interactions.
Results:
While treatment with hFIX-mFc altered the early development of anti-FIX IgG, no significant differences in anti-FIX antibody titers were observed at the end of the treatment regimen (5 weeks) or upon anamnestic response (5 months). However, treatment with hFIX-mFc elicited higher FIX-neutralizing antibody levels and resulted in reduced IgE titers compared to the hFIX-treated group. Additionally, differences in plasma cytokine levels and in vitro CD4(+) T cell responses suggest that while hFIX treatment triggered a Th2-biased immune response; hFIX-mFc treatment induced Th1-biased CD4(+) T cells. We also show that hFIX-mFc bound to soluble FcγRs and engaged with FcγRs on different cell types, which may impact antigen presentation.
Conclusions:
These studies provide a model system to study how Fc-fusion proteins may affect immune mechanisms. We used this model to demonstrate a plausible mechanism by which Fc-fusion may modulate the IgE response to hFIX. This model may be appropriate for investigating the rare but severe IgE-mediated anaphylaxis reaction to hFIX infusions in HB patients. This article is protected by copyright. All rights reserved.
The increasing diversity of influenza strains circulating in swine herds escalates the potential for the emergence of novel pandemic viruses and highlights the need for swift development of new vaccines. Baculovirus has proven to be a flexible platform for the generation of recombinant forms of hemagglutinin (HA) including subunit, VLP-displayed, and baculovirus-displayed antigens. These presentations have been shown to be efficacious in mouse, chicken, and ferret models but little is known about their immunogenicity in pigs. To assess the utility of these HA presentations in swine, Baculovirus constructs expressing HA fused to swine IgG2a Fc, displayed in a FeLV gag VLP, or displayed in the baculoviral envelope were generated. Vaccines formulated with these antigens were administered to groups of pigs who were subsequently challenged with H1α cluster H1N1 swine influenza virus (SIV) A/Swine/Indiana/1726/88. Our results demonstrate that vaccination with any of these three vaccines elicits robust hemagglutinin inhibition titers in the serum and decreased the severity of SIV-associated lung lesions after challenge when compared to placebo-vaccinated controls. In addition, the number of pigs with virus detected in the lungs and nasal passages was reduced. Taken together, the results demonstrate that these recombinant approaches expressed with the baculovirus expression vector system may be viable options for development of SIV vaccines for swine.
Worldwide spreading of H5 and H7 highly pathogenic influenza viruses of the avian origin, which periodically infect and kill humans without prior adaptation, poses a constant threat of the new pandemic. The effectiveness of the pandemic prevention completely depends on the quality of the existing influenza vaccines. Typical methods of the vaccine production from the antigenically relevant strains are problematic in case of high virulent H5 and H7 viruses. Therefore, new approaches to the construction of the vaccine strains and production technologies are required in order to protect the population.
Current diagnostic tests for tick-borne encephalitis virus (TBEV) infections require high biosafety facilities for antigen preparation, and can cross-react with other flaviviruses. There is therefore a need to develop safe, inexpensive serodiagnostic tools with high specificity and sensitivity. In this study, a recombinant plasmid that expresses the membrane (prM) and envelope (E) proteins of TBEV fused to the Fc domain of rabbit IgG was constructed and expressed in mammalian cells. The E-Fc proteins were secreted as soluble homodimers, which retained reactivity with anti-TBEV and anti-rabbit IgG antibodies. The E-Fc proteins were then used to develop an enzyme-linked immunosorbent assay (ELISA) to detect TBEV antibodies in rodent and human sera. Compared with the neutralization test, the ELISA had over 90% sensitivity and specificity. In addition, the assay showed no cross reactivity with Japanese encephalitis antibodies. These findings suggest that the E-Fc ELISA may be a useful tool for TBEV serodiagnosis.
Traditional vaccines such as inactivated or live attenuated vaccines, are gradually giving way to more biochemically defined vaccines that are most often based on a recombinant antigen known to possess neutralizing epitopes. Such vaccines can offer improvements in speed, safety and manufacturing process but an inevitable consequence of their high degree of purification is that immunogenicity is reduced through the lack of the innate triggering molecules present in more complex preparations. Targeting recombinant vaccines to antigen presenting cells (APCs) such as dendritic cells however can improve immunogenicity by ensuring that antigen processing is as efficient as possible. Immune complexes, one of a number of routes of APC targeting, are mimicked by a recombinant approach, crystallizable fragment (Fc) fusion proteins, in which the target immunogen is linked directly to an antibody effector domain capable of interaction with receptors, FcR, on the APC cell surface. A number of virus Fc fusion proteins have been expressed in insect cells using the baculovirus expression system and shown to be efficiently produced and purified. Their use for immunization next to non-Fc tagged equivalents shows that they are powerfully immunogenic in the absence of added adjuvant and that immune stimulation is the result of the Fc-FcR interaction. © 2012 Springer Science+Business Media Dordrecht. All rights are reserved.
Arguably, vaccination represents the single most effective medical intervention ever developed. Yet, vaccines have failed to provide any or adequate protection against some of the most significant global diseases. The pathogens responsible for these vaccine-recalcitrant diseases have properties that allow them to evade immune surveillance and misdirect or eliminate the immune response. However, genomic and systems biology tools, novel adjuvants and delivery systems, and refined molecular insight into protective immunity have started to redefine the landscape, and results from recent efficacy trials of HIV and malaria vaccines have instilled hope that another golden age of vaccines may be on the horizon.
Strategies to prevent the sexual transmission of HIV include vaccines that elicit durable, protective mucosal immune responses.
A key to effective mucosal immunity is the capacity for antigens administered locally to cross epithelial barriers. Given
the role of neonatal Fc receptor (FcRn) in transferring IgG across polarized epithelial cells which line mucosal surfaces,
FcRn might be useful for delivering HIV vaccine antigens across mucosal epithelial barriers to the underlying antigen-presenting
cells. Chimeric proteins composed of HIV Gag (p24) fused to the Fc region of IgG (Gag-Fc) bind efficiently to airway mucosa
and are transported across this epithelial surface. Mice immunized intranasally with Gag-Fc plus CpG adjuvant developed local
and systemic immunity, including durable B and T cell memory. Gag-specific immunity was sufficiently potent to protect against
an intravaginal challenge with recombinant vaccinia virus expressing the HIV Gag protein. Intranasal administration of a Gag-Fc/CpG
vaccine protected at a distal mucosal site. Our data suggest that targeting of FcRn with chimeric immunogens may be an important
strategy for mucosal immunization and should be considered a new approach for preventive HIV vaccines.
The rapid genetic drift of influenza virus hemagglutinin is an obstacle to vaccine efficacy. Previously, we found that the consensus hemagglutinin DNA vaccine (pCHA5) can only elicit moderate neutralization activities toward the H5N1 clade 2.1 and clade 2.3 viruses. Two approaches were thus taken to improve the protection broadness of CHA5. The first one was to include certain surface amino acids that are characteristic of clade 2.3 viruses to improve the protection profiles. When we immunized mice with CHA5 harboring individual mutations, the antibodies elicited by CHA5 containing P157S elicited higher neutralizing activity against the clade 2.3 viruses. Likewise, the viruses pseudotyped with hemagglutinin containing 157S became more susceptible to neutralization. The second approach was to update the consensus sequence with more recent H5N1 strains, generating a second-generation DNA vaccine pCHA5II. We showed that pCHA5II was able to elicit higher cross-neutralization activities against all H5N1 viruses. Comparison of the neutralization profiles of CHA5 and CHA5II, and the animal challenge studies, revealed that CHA5II induced the broadest protection profile. We concluded that CHA5II combined with electroporation delivery is a promising strategy to induce antibodies with broad cross-reactivities against divergent H5N1 influenza viruses.
Almost all infectious diseases are initiated at mucosal surfaces, yet intramuscular or subcutaneous vaccination usually provides only minimal protection at sites of infection owing to suboptimal activation of the mucosal immune system. The neonatal Fc receptor (FcRn) mediates the transport of IgG across polarized epithelial cells lining mucosal surfaces. We mimicked this process by fusing a model antigen, herpes simplex virus type-2 (HSV-2) glycoprotein gD, to an IgG Fc fragment. Intranasal immunization, together with the adjuvant CpG, completely protected wild-type, but not FcRn knockout, mice after intravaginal challenge with virulent HSV-2 186. This immunization strategy induced efficient mucosal and systemic antibody, B- and T-cell immune responses, with stable protection for at least 6 months after vaccination in most of the immunized animals. The FcRn-IgG transcellular transport pathway may provide a general delivery route for subunit vaccines against many mucosal pathogens.
The hemagglutinins (HAs) of human H1 and H3 influenza viruses and avian H5 influenza virus were produced as recombinant fusion
proteins with the human immunoglobulin Fc domain. Recombinant HA-human immunoglobulin Fc domain (HA-HuFc) proteins were secreted
from baculovirus-infected insect cells as glycosylated oligomer HAs of the anticipated molecular mass, agglutinated red blood
cells, were purified on protein A, and were used to immunize mice in the absence of adjuvant. Immunogenicity was demonstrated
for all subtypes, with the serum samples demonstrating subtype-specific hemagglutination inhibition, epitope specificity similar
to that seen with virus infection, and neutralization. HuFc-tagged HAs are potential candidates for gene-to-vaccine approaches
to influenza vaccination.
Cellular Fcγ receptors are essential for IgG-dependent effector functions in vivo. There is convincing evidence that selective activating Fcγ receptors are responsible for the activity of individual IgG subclasses. Thus, IgG1 activity is absent in FcγRIII-deficient mice, and several studies suggest that the activity of the most potent IgG subclasses, IgG2a and IgG2b, might be dependent on either individual or a combination of activating FcγRs. To study the role of individual activating FcγRs for IgG subclass activity, we generated an FcγRIV-deficient mouse and showed that a variety of IgG2a- and IgG2b-dependent effector functions are impaired in the absence of this activating Fc receptor in models of autoimmunity and antibody-dependent cellular cytotoxicity.
The survival of long-lived plasma cells, which produce most serum immunoglobulin, is central to humoral immunity. We found here that the inhibitory Fc receptor FcgammaRIIb was expressed on plasma cells and controlled their persistence in the bone marrow. Crosslinking FcgammaRIIb induced apoptosis of plasma cells, which we propose contributes to the control of their homeostasis and suggests a method for therapeutic deletion. Plasma cells from mice prone to systemic lupus erythematosus did not express FcgammaRIIb and were protected from apoptosis. Human plasmablasts expressed FcgammaRIIb and were killed by crosslinking, as were FcgammaRIIb-expressing myeloma cells. Our results suggest that FcgammaRIIb controls bone marrow plasma cell persistence and that defects in it may contribute to autoantibody production.
Preclinical tests of therapeutic antibodies are frequently carried out in mice to evaluate pharmacokinetics and efficacy. However, the observation that mouse IgG are cleared rapidly from the human circulation suggests that mice may not always be an ideal model. The Fc receptor, FcRn, regulates the serum half-lives of IgG in mice and most likely has a similar function in humans. In the current study we have carried out an extensive analysis of the interaction of the human or mouse forms of FcRn with IgG from various species using surface plasmon resonance. We show that in contrast to mouse FcRn, human FcRn is surprisingly stringent in its binding specificity for IgG derived from different species. Human FcRn binds to human, rabbit and guinea pig IgG, but not significantly to rat, bovine, sheep or mouse IgG (with the exception of weak binding to mouse IgG2b). In contrast, mouse FcRn binds to all IgG analyzed. The lack of binding of human FcRn to mouse IgG1 has been confirmed using transfectants that have been engineered to express human FcRn on the cell surface. Our results provide a molecular explanation for the enigmatic observation that mouse IgG behave anomalously in humans. These studies have implications for the successful application of therapeutic antibodies.
Four murine IgG subclasses display markedly different Fc-associated effector functions because of their differential binding to three activating IgG Fc receptors (FcgammaRI, FcgammaRIII, and FcgammaRIV) and C1q. Previous analysis of IgG subclass switch variants of 34-3C anti-RBC monoclonal autoantibodies revealed that the IgG1 subclass, which binds only to FcgammaRIII and fails to activate complement, displayed the poorest pathogenic potential. This could be related to the presence of a three amino acid deletion at positions 233-235 in the CH2 domain uniquely found in this subclass. To address this question, IgG1 insertion and IgG2b deletion mutants at positions 233-235 of 34-3C anti-RBC Abs were generated, and their ability to initiate effector functions and their pathogenicity were compared with those of the respective wild-type Abs. The insertion of amino acid residues at positions 233-235 enabled the IgG1 subclass to bind FcgammaRIV but did not improve the binding to C1q. Accordingly, its pathogenicity was enhanced but still inferior to that of IgG2b. In contrast, the IgG2b deletion mutant lost its ability to bind to FcgammaRIV and activate complement. Consequently, its pathogenicity was markedly diminished to a level comparable to that of IgG1. Our results demonstrated that the initiation of FcgammaR- and complement-mediated effector functions of IgG2b was profoundly affected by the three amino acid deletion at positions 233-235, but that this natural three amino acid deletion could only partially explain the poor binding of IgG1 to FcgammaRIV and C1q. This indicates the lack in the IgG1 subclass of as yet unknown motifs promoting efficient interaction with FcgammaRIV and C1q.
H5N1 influenza viruses have spread extensively among wild birds and domestic poultry. Cross-species transmission of these viruses to humans has been documented in over 380 cases, with a mortality rate of approximately 60%. There is great concern that a H5N1 virus would acquire the ability to spread efficiently between humans, thereby becoming a pandemic threat. An H5N1 influenza vaccine must, therefore, be an integral part of any pandemic preparedness plan. However, traditional methods of making influenza vaccines have yet to produce a candidate that could induce potently neutralizing antibodies against divergent strains of H5N1 influenza viruses. To address this need, we generated a consensus H5N1 hemagglutinin (HA) sequence based on data available in early 2006. This sequence was then optimized for protein expression before being inserted into a DNA plasmid (pCHA5). Immunizing mice with pCHA5, delivered intramuscularly via electroporation, elicited antibodies that neutralized a panel of virions that have been pseudotyped with the HA from various H5N1 viruses (clades 1, 2.1, 2.2, 2.3.2, and 2.3.4). Moreover, immunization with pCHA5 in mice conferred complete (clades 1 and 2.2) or significant (clade 2.1) protection from H5N1 virus challenges. We conclude that this vaccine, based on a consensus HA, could induce broad protection against divergent H5N1 influenza viruses and thus warrants further study.
We engineered an antibody expressing in the third complementarity-determining region of its heavy chain variable region a "foreign" epitope, the repetitive tetrapeptide Asn-Ala-Asn-Pro (NANP) of the circumsporozoite protein of Plasmodium falciparum parasite, one of the etiologic agents of malaria in humans. A monoclonal antibody to P. falciparum specific for the (NANP)n amino acid sequence bound to the engineered antibody, and a synthetic (NANP)3 peptide blocked this interaction. Immunization of rabbits and mice with the engineered antibody resulted in the elicitation of a humoral response to (NANP)3 synthetic peptide and P. falciparum parasite. In mice, in which immunity to the (NANP)n epitope is highly restricted by immune response genes, antibodies were induced in responder and nonresponder haplotypes of the major histocompatibility complex. Rabbit antibodies efficiently inhibited the in vitro invasion of cultured liver cells by P. falciparum parasite. Collectively, this study indicates that immunity to malaria in the absence of the parasite can be induced using antibody variable regions engineered to mimic the parasite's molecular structure. In general terms, the results suggest that antibody (idiotype) mimicry of an exogenous antigen is possible and may only require a discrete stretch of identity between the two molecules. The implication for the preparation of antibody-based vaccines and idiotype regulation of immunity are discussed.
A newly-constructed antibody-like molecule containing the gp120-binding domain of the receptor for human immunodeficiency virus blocks HIV-1 infection of T cells and monocytes. Its long plasma half-life, other antibody-like properties, and potential to block all HIV isolates, make it a good candidate for therapeutic use.
A major pathway in the clearance of pathogens involves the coating of the pathogen with specific antibodies, and the binding of the antibody Fc region to cell receptors. This can trigger engulfment of the pathogen by phagocytes or lysis by killer cells. By oligonucleotide site-directed mutagenesis we have engineered a single amino acid change in a mouse IgG2b antibody (Glu 235----Leu) which now enables the antibody to bind to the FcRI (high affinity) receptor on human monocytes with a 100-fold improvement in affinity. This indicates that Leu 235 is a major determinant in the binding of antibody to FcRI and that the receptor may interact directly with the region linking the CH2 domain to the hinge. Tailoring the affinity of antibodies for cell receptors could help dissect their role in clearing pathogen.
HIV-1 vpr encodes a 96-amino acid, nuclear protein whose function is not well understood. Unlike the other lentivirus regulatory proteins, Vpr is present in virions at relatively high copy number. In cells, Vpr is localized to the nucleus. Possible functions for vpr consistent with these findings include the nuclear import of preintegration complexes, transactivation of cellular genes, or induction of cellular differentiation. We show here, using both replication competent, macrophage-tropic virus and a sensitive, single-cycle luciferase HIV-1 reporter vector, that vpr is important for efficient viral replication in primary monocyte/macrophages, but appears to play no role in activated or resting T cell infection. The block to infection in monocytes was localized by PCR analysis of newly synthesized viral DNA and with the luciferase reporter vector to a stage in the viral life cycle after entry and reverse transcription, yet prior to, or at the time of, proviral transcription. In addition, infection of mononuclear phagocytes with virions that had been loaded with Vpr molecules in the producer cells by trans-complementation still showed a vpr-phenotype. These data suggest a role for vpr molecules produced in newly infected cells, in addition to its presumed function in the virion.
To test the hypothesis that blockade of B7-triggered costimulation by donor cells could preclude allograft rejection, we coated crude islet allograft preparations in vitro for 1 h with a murine CTLA4/Fc fusion protein. Murine CTLA4/Fc blocks the proliferative response in primary mixed lymphocyte cultures (MLC) and Con A-stimulated murine spleen cell cultures by 85 to 95%. Responder cells from a primary MLC containing mCTLA4/Fc were hyporesponsive upon restimulation to the same stimulator cells in a secondary MLC lacking mCTLA4/Fc. Because of mutations in the Fc gamma RI and C'1q binding sites of the Fc portion of the murine CTLA4/Fc fusion protein, the molecule binds to, but does not target, cells for Ab-dependent cellular cytotoxicity or complement-directed cytolysis. Although systemic immunosuppression was not applied, 42% (10 of 24) of B6AF1 recipients of islet allografts pretreated with CTLA4/Fc were permanently engrafted. Further, 50% of hosts bearing functioning islet allografts more than 150 days post-transplant were formally proved to be tolerant to donor tissues. A persistent CD4+ and CD8+ T cell infiltrate surrounding, but not invading, islet grafts in tolerant hosts was discerned. In control experiments, 89% (8 of 9) of islet allografts coated with mIgG3, and 100% (n = 10) pretreated with media alone were rejected. Thus, we conclude that 1) B7-triggered costimulation by donor APCs is an important element of rejection, and 2) blockade of the B7 pathway by in vitro allograft manipulation is able to induce tolerance.
Several prior reports have identified peptides that are naturally associated with major histocompatibility complex (MHC) class II molecules on presenting cells. We have examined the delivery of a peptide from exogenous sources to MHC class II molecules. The peptide derives from the influenza virus hemagglutinin (HA) and activates a CD4+ T cell hybridoma. In functional assays of antigen presentation, this epitope is delivered effectively to T cells either in the context of influenza virus or chimeric immunoglobulin (Ig) molecules (Ig-HA) in which the peptide has replaced the CDR3 loop of the heavy chain. We find that the identical 11-mer peptide can be isolated from mouse MHC class II antigens whether the exogenous source of peptide is free HA peptide, the Ig-HA chimera, or ultraviolet-inactivated PR8 influenza virus. The Ig-HA chimera proves to be the most efficient vehicle for charging class II molecules via the exogenous route. Given the fact that self Igs represent natural long-lived carriers, we suggest that antigenized Igs have considerable potential for peptide delivery to MHC molecules in situ.
Using the process of "antibody antigenization," we engineered two antibody molecules carrying in the third complementarity-determining region of the heavy chain variable domain a 7-mer or a 15-mer peptide epitope of the first extracellular domain (D1) of human CD4 receptor--namely, Ser-Phe-Leu-Thr-Lys-Gly-Pro-Ser (SFLTKGPS; positions 42 through 49) and Gly-Ser-Phe-Leu-Thr-Lys-Gly-Pro-Ser-Lys-Leu-Asn-Asp-Arg-Ala (GSFLTKGPSKLNDRA; positions 41 through 55). These amino acid sequences are contained in the consensus binding site for the human immunodeficiency virus (HIV) on CD4 receptor. Both antigenized antibodies (AgAbs) bound recombinant gp120 and were recognized by a prototype monoclonal antibody to CD4 whose binding site is within amino acid residues 41-55. AgAbs were then used as immunogens in rabbits and mice to elicit a humoral response against CD4. Only the AgAb carrying the sequence 41GSFLTKGPSKLN-DRA55 induced a response against CD4. The induced antibodies showed specificity for the amino acid sequence of CD4 engineered in the AgAb molecule, were able to inhibit the formation of syncytia between human CD4+ T cells MOLT-3 and 8E5 (T cells that are constitutively infected with HIV), and stained human CD4+ CEM T cells. Four murine monoclonal antibodies were used to analyze the relationship between syncytia inhibition and CD4 binding at the single antibody level, and indicated that recognition of native CD4 is not an absolute requirement for inhibition of syncytia. This study demonstrates that antigenized antibodies can be used as immunogens to elicit site-specific and biologically active immunity to CD4. The importance of this approach as a general way to induce anti-receptor immunity and as a possible new measure to immunointervention in HIV infection is discussed.
Oral administration of rabbit secretory IgA (sIgA) to adult BALB/c mice induced IgA+, IgM+, and IgG+ lymphoblasts in the Peyer's patches, whose fusion with myeloma cells resulted in hybridomas producing IgA, IgM, and IgG1 antibodies to the secretory component (SC). This suggests that SC could serve as a vector to target protective epitopes into mucosal lymphoid tissue and elicit an immune response. We tested this concept by inserting a Shigella flexneri invasin B epitope into SC, which, following reassociation with IgA, was delivered orally to mice. To identify potential insertion sites at the surface of SC, we constructed a molecular model of the first and second Ig-like domains of rabbit SC. A surface epitope recognized by an SC-specific antibody was mapped to the loop connecting the E and F beta strands of domain I. This 8-amino acid sequence was replaced by a 9-amino acid linear epitope from S. flexneri invasin B. We found that cellular trafficking of recombinant SC produced in mammalian CV-1 cells was drastically altered and resulted in a 50-fold lower rate of secretion. However, purification of chimeric SC could be achieved by Ni2+-chelate affinity chromatoraphy. Both wild-type and chimeric SC bound to dimeric IgA, but not to monomeric IgA. Reconstituted sIgA carrying the invasin B epitope within the SC moiety triggers the appearance of seric and salivary invasin B-specific antibodies. Thus, neo-antigenized sIgA can serve as a mucosal vaccine delivery system inducing systemic and mucosal immune responses.
T cell receptors on CD4(+) lymphocytes recognize antigen-derived peptides presented by major histocompatibility complex (MHC) class II molecules. A very limited set of peptides among those that may potentially bind MHC class II is actually presented to T lymphocytes. We here examine the role of two receptors mediating antigen internalization by antigen presenting cells, type IIb2 and type III receptors for IgG (FcgammaRIIb2 and FcgammaRIII, respectively), in the selection of peptides for presentation to T lymphocytes. B lymphoma cells expressing recombinant FcgammaRIIb2 or FcgammaRIII were used to assess the presentation of several epitopes from two different antigens. 4 out of the 11 epitopes tested were efficiently presented after antigen internalization through FcgammaRIIb2 and FcgammaRIII. In contrast, the 7 other epitopes were efficiently presented only when antigens were internalized through FcgammaRIII, but not through FcgammaRIIb2. The capacity to present these latter epitopes was transferred to a tail-less FcgammaRIIb2 by addition of the FcgammaRIII-associated gamma chain cytoplasmic tail. Mutation of a single leucine residue at position 35 of the gamma chain cytoplasmic tail resulted in the selective loss of presentation of these epitopes. Therefore, the nature of the receptor that mediates internalization determines the selection of epitopes presented to T lymphocytes within single protein antigens.
Autoantibodies and immune complexes are major pathogenic factors in autoimmune injury, responsible for initiation of the inflammatory cascade and its resulting tissue damage. This activation results from the interaction of immunoglobulin (Ig)G Fc receptors containing an activation motif (ITAM) with immune complexes (ICs) and cytotoxic autoantibodies which initiates and propagates an inflammatory response. In vitro, this pathway can be interrupted by coligation to FcgammaRIIB, an IgG Fc receptor containing an inhibitory motif (ITIM). In this report, we describe the in vivo consequences of FcgammaRII deficiency in the inflammatory response using a mouse model of IC alveolitis. At subthreshold concentrations of ICs that fail to elicit inflammatory responses in wild-type mice, FcgammaRII-deficient mice developed robust inflammatory responses characterized by increased hemorrhage, edema, and neutrophil infiltration. Bronchoalveolar fluids from FcgammaRII-/- stimulated mice contain higher levels of tumor necrosis factor and chemotactic activity, suggesting that FcgammaRII deficiency lowers the threshold of IC stimulation of resident cells such as the alveolar macrophage. In contrast, complement- and complement receptor-deficient mice develop normal inflammatory responses to suprathreshold levels of ICs, while FcRgamma-/- mice are completely protected from inflammatory injury. An inhibitory role for FcgammaRII on macrophages is demonstrated by analysis of FcgammaRII-/- macrophages which show greater phagocytic and calcium flux responses upon FcgammaRIII engagement. These data reveal contrasting roles for the cellular receptors for IgG on inflammatory cells, providing a regulatory mechanism for setting thresholds for IC sensitivity based on the ratio of ITIM to ITAM FcgammaR expression. Exploiting the FcgammaRII inhibitory pathway could thus provide a new therapeutic approach for modulating antibody-triggered inflammation.
Dendritic cells (DCs) express several receptors for the Fc portion of immunoglobulin (Ig)G (FcγR), which mediate internalization
of antigen–IgG complexes (immune complexes, ICs) and promote efficient major histocompatibility complex (MHC) class II–restricted
antigen presentation. We now show that FcγRs have two additional specific attributes in murine DCs: the induction of DC maturation
and the promotion of efficient MHC class I–restricted presentation of peptides from exogenous, IgG-complexed antigens. Both
FcγR functions require the FcγR-associated γ chain. FcγR-mediated MHC class I–restricted antigen presentation is extremely
sensitive and specific to immature DCs. It requires proteasomal degradation and is dependent on functional peptide transporter
associated with antigen processing, TAP1-TAP2. By promoting DC maturation and presentation on both MHC class I and II molecules,
ICs should efficiently sensitize DCs for priming of both CD4+ helper and CD8+ cytotoxic T lymphocytes in vivo.
Immunization with IgG/Ag or IgE/Ag complexes leads to a higher production of specific Abs than immunization with Ag alone. The enhancing effect of IgE is exclusively dependent upon the low-affinity receptor for IgE, Fc epsilon RII, whereas the mechanism behind IgG-mediated enhancement is unknown. We have investigated whether receptors for the Fc part of IgG are required for responses to IgG/Ag. Mice lacking the gamma subunit of Fc receptors (FcRs) (FcR gamma-/-), Fc gamma RII (Fc gamma RII-/-), or Fc gamma RIII (Fc gamma RIII-/-) were immunized with BSA-2,4,6-trinitrophenyl (TNP) alone or BSA-TNP complexed to monoclonal TNP-specific IgG1, IgG2a, or IgG2b. As expected, all subclasses enhanced the Ab-response to BSA in wild-type mice. Enhancement was in the same order of magnitude in Fc gamma RIII-/- mice (</=177-fold of controls administered Ag alone), whereas it was abrogated in FcR gamma-/- mice and augmented in Fc gamma RII-/- mice (</=5147-fold of controls). The response to IgE/Ag complexes in FcR gamma-/- and Fc gamma RII-/- mice was similar to that se for wild-type mice, demonstrating that non-Fc gamma R-dependent responses were normal. Our observations suggest that IgG/Ag complexes enhance Ab responses via Fc gamma Rs. Moreover, they reveal a strong negative regulation of Ab responses to IgG/Ag exerted by Fc gamma RII.
Novel tuberculosis DNA vaccines encoding native ESAT-6, MPT-64, KatG, or HBHA mycobacterial proteins or the same proteins fused to tissue plasminogen activator (TPA) signal sequences were evaluated for their capacity to elicit humoral, cell-mediated, and protective immune responses in vaccinated mice. While all eight plasmids induced specific humoral responses, the constructs expressing the TPA fusions generally evoked higher antibody responses in vaccinated hosts. Although most of the DNA vaccines tested induced a substantial gamma interferon response in the spleen, the antigen-specific lung responses were 2- to 10-fold lower than the splenic responses at the time of challenge. DNA vaccines encoding the ESAT-6, MPT-64, and KatG antigens fused to TPA signal sequences evoked significant protective responses in mice aerogenically challenged with low doses of Mycobacterium tuberculosis Erdman 17 to 21 days after the final immunization. However, the protective response induced by live Mycobacterium bovis BCG vaccine was greater than the response induced by any of the DNA vaccines tested. These results suggest that the tuberculosis DNA vaccines were able to elicit substantial immune responses in suitably vaccinated mice, but further refinements to the constructs or the use of alternative immunization strategies will be needed to improve the efficacy of these vaccine candidates.
Generation of the B cell recall response appears to involve interaction of Ag, in the form of an immune complex (IC) trapped on follicular dendritic cells (FDCs), with germinal center (GC) B cells. Thus, the expression of receptors on FDC and B cells that interact with ICs could be critical to the induction of an optimal recall response. FDCs in GCs, but not in primary follicles, express high levels of the IgG Fc receptor Fc gamma RIIB. This regulated expression of Fc gamma RIIB on FDC and its relation to recall Ab responses were examined both in vitro and in vivo. Trapping of IC in spleen and lymph nodes of Fc gamma RII-/- mice was significantly reduced compared with that in wild-type controls. Addition of ICs to cultures of Ag-specific T and B cells elicited pronounced Ab responses only in the presence of FDCs. However, FDCs derived from Fc gamma RIIB-/- mice supported only low level Ab production in this situation. Similarly, when Fc gamma RIIB-/- mice were transplanted with wild-type Ag-specific T and B cells and challenged with specific Ag, the recall responses were significantly depressed compared with those of controls with wild-type FDC. These results substantiate the hypothesis that FcgammaRIIB expression on FDCs in GCs is important for FDCs to retain ICs and to mediate the conversion of ICs to a highly immunogenic form and for the generation of strong recall responses.
Immunoglobulins (Igs) are heterodimeric proteins consisting of two heavy (H) and two light (L) chains, each of which encodes a V domain and one or more C domains. The V domain is the product of a complex series of gene rearrangement events that endow Igs, as a population, to recognize an almost unlimited array of self and nonself antigens. The H chain C domains encode effector functions that can include activation of complement, binding to Fc receptors, and inducing or maintaining inflammation.
Several prior reports have identified peptides that are naturally associated with major histocompatibility complex (MHC) class II molecules on presenting cells. We have examined the delivery of a peptide from exogenous sources to MHC class II molecules. The peptide derives from the influenza virus hemagglutinin (HA) and activates a CD4+ T cell hybridoma. In functional assays of antigen presentation, this epitope is delivered effectively to T cells either in the context of influenza virus or chimeric immunoglobulin (Ig) molecules (Ig-HA) in which the peptide has replaced the CDR3 loop of the heavy chain. We find that the identical 11-mer peptide can be isolated from mouse MHC class II antigens whether the exogenous source of peptide is free HA peptide, the Ig-HA chimera, or ultraviolet-inactivated PR8 influenza virus. The Ig-HA chimera proves to be the most efficient vehicle for charging class II molecules via the exogenous route. Given the fact that self Igs represent natural long-lived carriers, we suggest that antigenized Igs have considerable potential for peptide delivery to MHC molecules in situ.
CD44 is a broadly distributed cell surface protein thought to mediate cell attachment to extracellular matrix components or specific cell surface ligands. We have created soluble CD44-immunoglobulin fusion proteins and characterized their reactivity with tissue sections and lymph node high endothelial cells in primary culture. The CD44 target on high endothelial cells is sensitive to enzymes that degrade hyaluronate, and binding of soluble CD44 is blocked by low concentrations of hyaluronate or high concentrations of chondroitin 4- and 6-sulfates. A mouse anti-hamster hyaluronate receptor antibody reacts with COS cells expressing hamster CD44 cDNA. In sections of all tissues examined, including lymph nodes and Peyer's patches, predigestion with hyaluronidase eliminated CD44 binding.
Germinal centers (GCs) are specialized microenvironments formed after infection where activated B cells can mutate their B-cell receptors to undergo affinity maturation. A stringent process of selection allows high affinity, non-self-reactive B cells to become long-lived memory B cells and plasma cells. While the precise mechanism of selection is still poorly understood, the last decade has advanced our understanding of the role of T cells and follicular dendritic cells (FDCs) in GC B-cell formation and selection. T cells and non-T-cell-derived CD40 ligands on FDCs are essential for T-dependent (TD) and T-independent GC formation, respectively. TD-GC formation requires Bcl-6-expressing T cells capable of signaling through SAP, which promotes formation of stable T:B conjugates. By contrast, differentiation of B blasts along the extrafollicular pathway is less dependent on SAP. T-follicular helper (Tfh) cell-derived CD40L, interleukin-21, and interleukin-4 play important roles in GC B-cell proliferation, survival, and affinity maturation. A role for FDC-derived integrin signals has also emerged: GC B cells capable of forming an immune synapse with FDCs have a survival advantage. This emerges as a powerful mechanism to ensure death of B cells that bind self-reactive antigen, which would not normally be presented on FDCs.
A need for antigen-processing and presentation to B cells is not widely appreciated. However, cross-linking of multiple B cell receptors (BCRs) by T-independent antigens delivers a potent signal that induces antibody responses. Such BCR cross-linking also occurs in germinal centers where follicular dendritic cells (FDCs) present multimerized antigens as periodically arranged antigen-antibody complexes (ICs). Unlike T cells that recognize antigens as peptide-MHC complexes, optimal B cell-responses are induced by multimerized FDC-ICs that simultaneously engage multiple BCRs. FDC-FcgammaRIIB mediates IC-periodicity and FDC-BAFF, FDC-IL-6 and FDC-C4bBP are co-stimulators. Remarkably, specific antibody responses can be induced by FDC-ICs in the absence of T cells, opening up the exciting possibility that people with T cell insufficiencies may be immunized with T-dependent vaccines via FDC-ICs.
FcgammaRIIB is the only inhibitory Fc receptor. It controls many aspects of immune and inflammatory responses, and variation in the gene encoding this protein has long been associated with susceptibility to autoimmune disease, particularly systemic lupus erythematosus (SLE). FcgammaRIIB is also involved in the complex regulation of defence against infection. A loss-of-function polymorphism in FcgammaRIIB protects against severe malaria, the investigation of which is beginning to clarify the evolutionary pressures that drive ethnic variation in autoimmunity. Our increased understanding of the function of FcgammaRIIB also has potentially far-reaching therapeutic implications, being involved in the mechanism of action of intravenous immunoglobulin, controlling the efficacy of monoclonal antibody therapy and providing a direct therapeutic target.
The human immunodeficiency virus type 1 (HIV-1) exploits the cell surface CD4 molecule to initiate the infection which can lead, eventually, to acquired immunodeficiency syndrome (AIDS). The HIV-1 envelope protein, gp120, interacts specifically with CD4 and soluble CD4 molecules have been shown to inhibit HIV infectivity in vitro. Effective inhibition in vivo may, however, require more potent reagents. We describe here the generation of molecules which combine the specificity of CD4 and the effector functions of different immunoglobulin subclasses. Replacing the VH and CH1 domains of either mouse gamma 2a or mu heavy chains with the first two N-terminal domains of CD4 results in molecules that are secreted in the absence of any immunoglobulin light chains. We find that the pentameric CD4-IgM chimaera is at least 1,000-fold more active than its dimeric CD4-IgG counterpart in syncytium inhibition assays and that effector functions, such as the binding of Fc receptors and the first component of the complement cascade (Clq), are retained. Similar chimaeric molecules, combining CD4 with human IgG were recently described by Capon et al., but these included the CH1 domain and did not bind Clq. Deletion of the CH1 domain may allow the association and secretion of heavy chains in the absence of light chains, and we suggest that the basic design of our constructs may be generally and usefully applied.
The elicitation of broadly cross-reactive HIV-1 neutralizing antibodies in humans remains a major challenge in developing a viable AIDS vaccine. We hypothesized that prolonged exposure to candidate vaccine immunogens could enhance the elicitation of such antibodies. In an attempt to develop HIV-1 vaccine immunogens with prolonged half-lives and increased stability, we constructed a fusion protein, gp41Fc, in which a truncated HIV-1 gp41(89.6) was fused to a human IgG(1) Fc. Gp41Fc is stable in solution, retains its antigenic structure and is highly immunogenic in rabbits. The serum titers reached 1:102,400 for the gp41Fc and 1:5,120 for gp140(89.6). Rabbit IgG neutralized diverse HIV-1 isolates and HIV-2, and the neutralization activity was attributed to gp41-specific IgG. The concentration of the gp41Fc in the serum correlated with the neutralization activity of rabbit IgG which recognized mostly conformation-independent epitopes on gp41 and predominantly bound to peptides derived from the gp41 immunodominant loop region. These results suggest that the prolonged half-life of gp41Fc in the serum may enhance the generation of cross-reactive neutralizing antibodies. Further research is needed to confirm and extend these results which may have implications for the development of vaccine immunogens with enhanced capability to elicit cross-reactive HIV-1-neutralizing antibodies.
Macrophages display remarkable plasticity and can change their physiology in response to environmental cues. These changes can give rise to different populations of cells with distinct functions. In this Review we suggest a new grouping of macrophage populations based on three different homeostatic activities - host defence, wound healing and immune regulation. We propose that similarly to primary colours, these three basic macrophage populations can blend into various other 'shades' of activation. We characterize each population and provide examples of macrophages from specific disease states that have the characteristics of one or more of these populations.
THE manufacture of predefined specific antibodies by means of permanent
tissue culture cell lines is of general interest. There are at present a
considerable number of permanent cultures of myeloma cells1,2
and screening procedures have been used to reveal antibody activity in
some of them. This, however, is not a satisfactory source of monoclonal
antibodies of predefined specificity. We describe here the derivation of
a number of tissue culture cell lines which secrete anti-sheep red blood
cell (SRBC) antibodies. The cell lines are made by fusion of a mouse
myeloma and mouse spleen cells from an immunised donor. To understand
the expression and interactions of the Ig chains from the parental
lines, fusion experiments between two known mouse myeloma lines were
carried out.
A new process, antigenization of antibodies, consisting of the expression of oligopeptides in the hypervariable loops of an antibody molecule is described. The potential applications of antigenized antibodies are discussed.
Molecular fusions of CD4, the receptor for human immunodeficiency virus (HIV), with immunoglobulin (termed CD4 immunoadhesins) possess both the gp120-binding and HIV-blocking properties of recombinant soluble CD4, and certain properties of IgG, notably long plasma half-life and Fc receptor binding. Here we show that a CD4 immunoadhesin can mediate antibody-dependent cell-mediated cytotoxicity (ADCC) towards HIV-infected cells, although, unlike natural anti-gp120 antibodies, it does not allow ADCC towards uninfected CD4-expressing cells that have bound soluble gp120 to the CD4 on their surface. In addition, CD4 immunoadhesin, like natural IgG molecules, is efficiently transferred across the placenta of a primate. These observations have implications for the therapeutic application of CD4 immunoadhesins, particularly in the area of perinatal transmission of HIV infection.
In humoral defence, pathogens are cleared by antibodies acting as adaptor molecules: they bind to antigen and trigger clearance mechanisms such as phagocytosis, antibody-dependent cell-mediated cytotoxicity and complement lysis. The first step in the complement cascade is the binding of C1q to the antibody. There are six heads on C1q, connected by collagen-like stems to a central stalk, and the isolated heads bind to the Fc portion of antibody rather weakly, with an affinity of 100 microM (ref. 3). Binding of antibody to multiple epitopes on an antigenic surface, aggregates the antibody and this facilitates the binding of several C1q heads, leading to an enhanced affinity of about 10 nM (ref. 1). Within the Fc portion of the antibody, C1q binds to the CH2 domain. The interaction is sensitive to ionic strength, and appears to be highly conserved throughout evolution as C1q reacts with IgG from different species (for example see ref. 8). By systematically altering surface residues in the mouse IgG2b isotype, we have localized the binding site for C1q to three side chains, Glu 318, Lys 320 and Lys 322. These residues are relatively conserved in other antibody isotypes, and a peptide mimic of this sequence is able to inhibit complement lysis. We propose that this sequence motif forms a common core in the interactions of IgG and C1q.
We determined the half-lives of several sets of murine monoclonal antibodies spanning all immunoglobulin isotypes in the serum. The antibodies in each set possess the same V region. With this approach, the differences in half-life observed between the different isotypes are independent of the V region carried by the monoclonal antibodies and therefore must relate to each other in the same way as the half-lives of each class of serum immunoglobulins.
The half-life of a monoclonal antibody of the γ2a isotype is identical to the average half-life of serum IgG2a, as previously determined (6-8 days; P. Vieira and K. Rajewsky, Eur. J. Immunol. 1986. 16: 871). Therefore, the half-lives determined with monoclonal antibodies possessing the same V region represent the half-life of the serum immunoglobulins.
In this way we calculated the half-life of IgM as 2 days, IgG3 and IgG1 as 6-8 days, IgG2b has a half-life of 4-6 days. IgE has a half-life of 12 h. A polymeric form of IgA was found to be eliminated from the serum with a half-life of 17-22 h.
To produce sufficient quantities of soluble T-cell receptor protein for detailed biochemical and biophysical analyses we have explored the use of immunoglobulin--T-cell receptor gene fusions. In this report we describe a chimeric gene construct containing a T-cell receptor alpha-chain variable (V) domain and the constant (C) region coding sequences of an immunoglobulin gamma 2a molecule. Cells transfected with the chimeric gene synthesize a stable protein product that expresses immunoglobulin and T-cell receptor antigenic determinants as well as protein A binding sites. We show that the determinant recognized by the anticlonotypic antibody A2B4.2 resides on the V alpha domain of the T-cell receptor. The chimeric protein associates with a normal lambda light chain to form an apparently normal tetrameric (H2L2, where H = heavy and L = light) immunoglobulin molecule that is secreted. Also of potential significance is the fact that a T-cell receptor V beta gene in the same construct is neither assembled nor secreted with the lambda light chain, and when expressed with a C kappa region it does not assemble with the chimeric V alpha C gamma 2a protein mentioned above. This indicates that not all T-cell receptor V regions are similar enough to immunoglobulin V regions for them to be completely interchangeable.
The concentration of IgG2a in the serum of C57BL/6 mice suppressed for the production of IgG2a was measured over a period of 3 months after suppression. The elimination of a minimum of 96% of the endogenous IgG2a molecules from the serum follows an exponential decay with a half-life of 6-8 days. A monoclonal IgG2a antibody injected into suppressed or normal mice is eliminated from the serum in the same fashion. We conclude that there are few, if any, "long-lived" IgG2a molecules present in the serum of C57BL/6 mice under physiological conditions.
Using monoclonal anti-trinitrophenyl (TNP) antibodies complexed to TNP-myoglobin-coated gold particles, we analysed at the ultrastructural level the retention by follicular dendritic cells (FDC) of immune complexes containing various antibody isotypes. Gold-labelled immune complexes were injected subcutaneously or intravenously into naive mice and, after 24 h, germinal centres of draining lymph nodes or spleen were examined by electron microscopy. FDC generally retained complexes containing IgG2a and IgG2b better than those formed with IgG1 or IgG3. IgM was rarely retained. FDC isolated from lymph nodes or spleens were incubated in vitro with gold-labelled complexes in a serum-free medium. IgG2a and IgG2b complexes were also retained in vitro in large quantities by FDC; IgG1 and IgG3 complexes were retained in smaller quantities or in highly variable quantities compared with IgG2; IgM complexes were rarely seen on FDC. There was no difference between FDC isolated from lymph nodes or from spleen with respect to the Ig isotypes required for Fc-mediated retention of immune complexes.
The hypervariable region 3 (V3) within the disulfide-bridged loop of the envelope protein of the human immunodeficiency virus type 1 (HIV-1) contains an amino acid sequence that was defined as a principal neutralizing determinant (PND). A 19-amino acid residue consensus sequence (designated V3C) predicted from the PND sequences of 245 isolates as well as a sequence from the PND of the WMJ2 HIV-1 isolate (designated V3M) were expressed on the variable region of murine-human immunoglobulin (Ig) chimeras that were designated Ig-V3C and Ig-V3M, respectively. The HIV-1 sequences on the Ig chimeras preserved their antigenicity and interacted with antibodies specific for peptides encompassing the V3C and V3M sequences. In baboons, Ig-V3C and Ig-V3M induced antibodies that bound V3C and V3M peptides as well as the glycoprotein gp120 envelope protein of HIV-1 MN isolate. In addition, the baboons' antisera were able to prevent infection of CD4 SupT1 susceptible T cells by HIV-1 MN. Finally, Ig-V3M chimeras were able to stimulate in vitro production of antibodies specific for the HIV-1 envelope-derived peptides by lymphocytes from HIV-1-infected human subjects.
The capacity to trap immune complexes (IC) was examined using purified peroxidase-antiperoxidase IgG on frozen sections of mouse spleen. In the absence of serum, binding of IC was confined to macrophages in the red pulp. However, when IC were applied in the presence of fresh mouse serum as a source of complement, trapping occurred on follicular dendritic cells (FDC) in the primary follicles. Trapping was specifically inhibited with monoclonal antibodies against complement receptor type 1 (CR1) (8C12) and CR 1/2 (7G6), but not with anti-CR3 (Mac-1). The binding mechanism of IC in the secondary follicles of animals immunized with sheep red blood cells differed from that observed in unimmunized mice. Here, trapping occurred both in the absence and presence of fresh mouse serum. In the absence of fresh mouse serum, trapping on FDC was mediated by FcR gamma II, as demonstrated by blocking with monoclonal antibody 2.4G2. FcR gamma II-mediated trapping was only observed on a relatively small proportion of FDC that was located in the light zone of the secondary follicles, while complement receptor-mediated IC trapping was observed in more expanding areas, including dark zone and corona. These results show that both complement receptor as well as Fc receptor can function in the binding of IC to FDC. They also provide evidence for the existence of functionally different FDC populations in the secondary lymphoid follicles of mouse spleen.
The immunogenicity of a soluble, non-self protein or peptide can be greatly enhanced by injecting this antigen coupled to an antibody specific for class II MHC molecules in the recipient. This adjuvant-independent immunization strategy is known as immunotargeting. We have investigated the ability of a mouse anti-class II MHC antibody to provide the three-dimensional framework for the reconstitution of a heterologous conformational B-cell epitope, specifically the A loop epitope from the influenza virus hemagglutinin (HA). From a panel of three anti-class II MHC immunoglobulin (Ig)-A loop constructs, we found that one of these, an insertion into the FR3 region of the Ig molecule, retained its specificity for mouse I-Ak. Although mouse monoclonal antibodies specific for the A loop region in the HA molecule were unable to react with the Ig-A loop variants, we did find that the heavy chain CDR3 insertion construct was able to elicit an A loop-specific, HA-reactive antibody response when used as an immunogen in rabbits. These results demonstrate the potential for the Ig molecule to function successfully as a structural framework for the reconstitution and presentation of heterologous conformational B-cell epitopes.
Three classes of Fc receptors for IgG, Fc gamma RI (CD64), Fc gamma RII (CD32), and Fc gamma RIII (CD16), are expressed on blood leukocytes. Although Fc gamma R are important phagocytic receptors on phagocytes, most reports suggest that dendritic cells lack Fc gamma R-mediated phagocytosis and express significant levels of only CD32. We now report that phagocytically active forms of both CD64 and CD32 are expressed significantly on at least one subset of human blood dendritic cells. Countercurrent elutriation and magnetic bead selection were used to rapidly enrich subsets of blood dendritic cells (CD33brightCD14-HLA-DRbrightCD83-) and monocytes (CD33brightCD14brightHLA-DRdimCD83-). Upon culture for 2 days, dendritic cells became CD83-positive and markedly increased HLA-DR expression, whereas monocytes did not express CD83 and exhibited reduced levels of HLA-DR. Constitutive CD64 expression was identified on this circulating dendritic cell population, but at a lower level than on monocytes. CD64 expression by dendritic cells and monocytes did not decrease during 2 days in culture, and was up-regulated on both cell types following incubation with IFN-gamma. Freshly isolated blood dendritic cells performed CD64- and CD32-mediated phagocytosis, although at a lower level than monocytes. Dendritic cells generated by culture of adherent mononuclear cells in granulocyte-macrophage CSF and IL-4 also up-regulated CD64 following IFN-gamma stimulation, and mediated CD64-dependent phagocytosis. These results indicate that both CD64 and CD32 expressed on blood dendritic cells may play a role in uptake of foreign particles and macromolecules through a phagocytic mechanism before trafficking to T cell-reactive areas.
The role of Ag-Ab complexes (or immune complexes; ICs) in the regulation of the maturation of the B cell immune response was investigated in mice perturbed in the deposition and retention of such complexes. Loss of surface expression of Fc gammaRI and Fc gammaRIII due to targeted disruption of the common FcR gamma-chain gene results in dramatically increased deposition of ICs on follicular dendritic cells (FDCs) in germinal centers (GCs), attributed to altered clearance of circulating ICs. Despite these changes in the trapping of ICs by FDCs, serum Ab production, V gene hypermutation, isotype class switching and Ab affinity maturation are overtly unaltered. Thus, substantially augmenting B cell cognate Ag density on FDCs does not alter the outcome of the maturation of the B cell response. The significance of this finding in terms of the currently accepted model for the generation of B cell memory is discussed.
Antibodies engineered in their variable domain to express epitopes of heterologous antigens-antigenized antibodies-function as immunogens. Only the third complementarity-determining region (CDR3) of the H chain has been used as the site of epitope expression, as this loop has the highest natural variability in length and amino acid composition. We demonstrate that the CDR2 can be engineered to express a 12-amino acid peptide, which is a T-cell determinant that enhances the response to a B-cell epitope peptide of Plasmodium falciparum expressed in the CDR3 of the same variable domain. Mice with this gene inoculated into the spleen mounted an antibody response against the B-cell epitope higher than mice receiving the gene coding for the B-cell epitope only. In vitro studies established that the two epitopes were independently immunogenic in vivo.
Inhibitory receptors have been proposed to modulate the in vivo cytotoxic response against tumor targets for both spontaneous and antibody-dependent pathways. Using a variety of syngenic and xenograft models, we demonstrate here that the inhibitory FcgammaRIIB molecule is a potent regulator of antibody-dependent cell-mediated cytotoxicity in vivo, modulating the activity of FcgammaRIII on effector cells. Although many mechanisms have been proposed to account for the anti-tumor activities of therapeutic antibodies, including extended half-life, blockade of signaling pathways, activation of apoptosis and effector-cell-mediated cytotoxicity, we show here that engagement of Fcgamma receptors on effector cells is a dominant component of the in vivo activity of antibodies against tumors. Mouse monoclonal antibodies, as well as the humanized, clinically effective therapeutic agents trastuzumab (Herceptin(R)) and rituximab (Rituxan(R)), engaged both activation (FcgammaRIII) and inhibitory (FcgammaRIIB) antibody receptors on myeloid cells, thus modulating their cytotoxic potential. Mice deficient in FcgammaRIIB showed much more antibody-dependent cell-mediated cytotoxicity; in contrast, mice deficient in activating Fc receptors as well as antibodies engineered to disrupt Fc binding to those receptors were unable to arrest tumor growth in vivo. These results demonstrate that Fc-receptor-dependent mechanisms contribute substantially to the action of cytotoxic antibodies against tumors and indicate that an optimal antibody against tumors would bind preferentially to activation Fc receptors and minimally to the inhibitory partner FcgammaRIIB.