Experimental evidence of pathogenic role of IgG autoantibodies in IgA nephropathy.

BACKGROUND: IgA nephropathy is thought to be an autoimmune disease wherein galactose-deficient IgA1 (Gd-IgA1) is recognized by IgG autoantibodies, resulting in formation and renal accumulation of nephritogenic immune complexes. Although this hypothesis is supported by recent findings that, in renal immunodeposits of IgA nephropathy patients, IgG is enriched for Gd-IgA1-specific autoantibodies, experimental proof is still lacking. METHODS: IgG isolated from sera of IgA nephropathy patients or produced as a recombinant IgG (rIgG) was mixed with human Gd-IgA1 to form immune complexes. IgG from healthy individuals served as a control. Nude and SCID mice were injected with human IgG and Gd-IgA1, in immune complexes or individually, and their presence in kidneys was ascertained by immunofluorescence. Pathologic changes in the glomeruli were evaluated by quantitative morphometry and exploratory transcriptomic profiling was performed by RNA-Seq. RESULTS: Immunodeficient mice injected with Gd-IgA1 mixed with IgG autoantibodies from patients with IgA nephropathy, but not Gd-IgA1 mixed with IgG from healthy individuals, displayed IgA, IgG, and mouse complement C3 glomerular deposits and mesangioproliferative glomerular injury with hematuria and proteinuria. Un-complexed Gd-IgA1 or IgG did not induce pathological changes. Moreover, Gd-IgA1-rIgG immune complexes injected into immunodeficient mice induced histopathological changes characteristic of human disease. Exploratory transcriptome profiling of mouse kidney tissues indicated that these immune complexes altered gene expression of multiple pathways, in concordance with the changes observed in kidney biopsies of patients with IgA nephropathy. CONCLUSIONS: This study provides the first in vivo evidence for a pathogenic role of IgG autoantibodies specific for Gd-IgA1 in the pathogenesis of IgA nephropathy.

CD22 and CD72 cooperatively contribute to the development of the reverse Arthus reaction model.

BACKGROUND: Local type III hypersensitivity reactions are acute inflammatory events induced by immune complex (IC) deposition. CD22 and CD72 are B cell-specific cell surface molecules that negatively regulate B cell function. OBJECTIVE: To elucidate the roles of CD22 and CD72 in the development of IgG-mediated type III hypersensitivity reactions. METHOD: The reverse Arthus reaction model in the skin was induced in mice lacking CD22 (CD22-/-), CD72 (CD72-/-), and both of them (CD22-/-/CD72-/-). Edema at 4h and hemorrhage at 8h after IC challenge were evaluated. Inflammatory cell infiltration and cytokine and chemokine expression were also examined. RESULTS: Edema and hemorrhage were significantly reduced in CD22-/-/CD72-/- mice compared with wild-type mice. The loss of both membrane proteins resulted in a greater decrease in edema at 4h, but not hemorrhage at 8h, than the loss of each protein alone. Infiltration of neutrophils, macrophages, and T cells, and the expression of TNF-α, IL-6, MIP-1α, and CCR5 mRNA were also diminished in the knockout mice compared to wild-type mice, and most significantly reduced in CD22-/-/CD72-/- mice. Regulatory T (Treg) cells in the spleen were significantly increased in all knockout mice at 4h. Significant differences in the severity of edema and hemorrhage between wild-type and knockout mice were lost when Treg cells were depleted in the knockout mice. CONCLUSION: These results demonstrate that CD22 and CD72 expression contribute to the development of the reverse Arthus reaction model and CD22 and CD72 might be therapeutic targets for human IC-mediated diseases.

Modulation of Antibody Responses to the V1V2 and V3 Regions of HIV-1 Envelope by Immune Complex Vaccines.

Prophylactic HIV vaccines must elicit antibodies (Abs) against the virus envelope glycoproteins (Env) to effectively prevent HIV infection. We investigated a vaccine platform that utilizes immune complexes made of Env proteins gp120 and monoclonal Abs (mAbs) against different gp120 epitopes. We previously observed alterations in V3 antigenicity upon formation of certain gp120/mAb complexes and demonstrated the ability of these complexes to modulate the elicitation of V3 Ab responses. However, the effects on the V1V2 domain, an important target for Abs that correlate with vaccine-induced protection against HIV, have not been studied, nor have immune complex vaccines made with non-B subtype Env. This study compared subtypes B (JRFL) and CRF_01.AE (A244) Env gp120 proteins in complex with selected gp120-specific mAbs. Allosteric and antigenic changes were detected on these immune complexes, indicating that gp120/mAb interaction induces alterations on the Env surface that may modify the Env immunogenic properties. To evaluate this idea, mice were immunized with gp120/mAb complexes or their uncomplexed gp120 counterparts. The overall serum IgG titers elicited against gp120 were comparable, but a marked skewing toward V1V2 or V3 was evident and dependent on the gp120 strain and the specificity of the mAb used to form the complexes. Compared with uncomplexed gp120JRFL, gp120JRFL complexed with CD4bs or V1V2 mAbs, but not with C2 or V3 mAbs, elicited V3 Abs of greater titers and breadth, and Abs more capable of neutralizing tier 1 virus. Epitope mapping revealed a shift to a more conserved site in the V3 crown. However, the complexes did not enhance V1V2 Ab response, and the elicited V1V2 Abs were not cross-reactive. This profile contrasts with Ab responses to gp120A244/mAb complexes. Notably, gp120A244/mAb complexes induced higher levels of V1V2 Abs with some cross-reactivity, while also stimulating weak or strain-specific V3 Abs. Sera from gp120A244/mAb complex-immunized animals displayed no measurable virus neutralization but did mediate Ab-dependent cellular phagocytosis, albeit at levels similar to that induced by gp120A244 alone. These data indicate the potential utility of immune complexes as vaccines to shape Ab responses toward or away from Env sites of interest.

Intracapillary immune complexes recruit and activate slan-expressing CD16+ monocytes in human lupus nephritis.

Lupus nephritis is a major cause of morbidity in patients with systemic lupus erythematosus. Among the different types of lupus nephritis, intracapillary immune complex (IC) deposition and accumulation of monocytes are hallmarks of lupus nephritis class III and IV. The relevance of intracapillary ICs in terms of monocyte recruitment and activation, as well as the nature and function of these monocytes are not well understood. For the early focal form of lupus nephritis (class III) we demonstrate a selective accumulation of the proinflammatory population of 6-sulfo LacNAc+ (slan) monocytes (slanMo), which locally expressed TNF-α. Immobilized ICs induced a direct recruitment of slanMo from the microcirculation via interaction with Fc γ receptor IIIA (CD16). Interestingly, intravenous immunoglobulins blocked CD16 and prevented cell recruitment. Engagement of immobilized ICs by slanMo induced the production of neutrophil-attracting chemokine CXCL2 as well as TNF-α, which in a forward feedback loop stimulated endothelial cells to produce the slanMo-recruiting chemokine CX3CL1 (fractalkine). In conclusion, we observed that expression of CD16 equips slanMo with a unique capacity to orchestrate early IC-induced inflammatory responses in glomeruli and identified slanMo as a pathogenic proinflammatory cell type in lupus nephritis.

Recombinant immune complexes as versatile and potent vaccines.

Immune complexes (IC) used as vaccines have the potential to enhance both antibody and cell-mediated immune responses over those obtained with antigen alone. However, difficulty of manufacture represents a significant hurdle to the widespread use of IC as vaccines. Recombinant IC (RIC) and their expression in plants enable manufacturing by the coordinate expression of immunoglobulin and antigen as a fusion protein. The use of a modular RIC system facilitates insertion of antigen genes and provides a broadly applicable platform that can be adapted for a wide variety of antigens.

A novel modified vaccination technique produces IgG antibodies that cause complement-mediated lysis of multiple myeloma cells carrying CD38 antigen.

Objectives were to: 1) induce a lytic IgG antibody (ab) response (via the so called `third vaccination method') against CD38 antigen (ag) residing on the extra-cellular domain of multiple myeloma (MM) cells in recipient rabbits, by combining the CD38 ag with donor-derived anti-CD38 ag lytic IgG ab into an immune complex (IC); and 2) determine whether abs produced would cause complement-mediated lysis (in vitro) of human MM cells containing CD38 ag. The vaccine was created in a two-step process. First, ab (rabbit anti-CD38 ag IgG ab) was raised in donor rabbits by injections of low molecular weight soluble CD38 ag in Freund's complete adjuvant (FCA) and aqueous solution. Second, transfer of pathogenic lytic IgG ab response into recipient rabbits was achieved by injections of ICs composed of CD38 ag and homologous anti-CD38 ag IgG ab. Consequently, recipient rabbits produced the same ab with the same specificity against the target ag as was present in the inoculum, namely agglutinating, precipitating and lytic (as demonstrated in vitro). In an in vitro study, in the presence of complement, donor and recipient rabbits' immune sera caused lysis of CD38 ag associated human MM cells. The most effective lytic ab response causing sera were those from donor rabbits injected with CD38 ag in FCA and those from rabbits injected with ICs, especially when they were administered in adjuvants. These results provided proof of concept that the third vaccination method has good potential as a stand-alone and efficacious method of controlling cancer.

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.

Intravenous IgA complexed with antigen reduces primary antibody response to the antigen and anaphylaxis upon antigen re-exposure by inhibiting Th1 and Th2 activation in mice.

CONTEXT: Serum IgG, IgE and IgM have been shown to enhance the primary antibody responses upon exposure to the soluble antigens recognized by those antibodies. However, how IgA affects these responses remains unknown. OBJECTIVE: We investigated the effects of intravenously administered monoclonal IgA on the immune responses in mice. MATERIALS AND METHODS: DBA/1J mice were immunized with ovalbumin in the presence or absence of anti-ovalbumin monoclonal IgA. The Th1 and Th2 immune responses to ovalbumin and the anaphylaxis induced by re-exposure to ovalbumin were measured. RESULTS: IgA complexed with antigen attenuated the primary antibody responses to the antigen in mice, in contrast to IgG2b and IgE. The primary antibody responses, i.e. the de novo synthesis of anti-ovalbumin IgG2a, IgG1 and IgE in the serum, and the subsequent anaphylaxis induced with re-exposure to ovalbumin were reduced by the co-injection of anti-ovalbumin monoclonal IgA at ovalbumin immunization. The Th1, Th2 and Tr1 cytokines interferon-γ, interleukin-4 and interleukin-10, respectively, released from ovalbumin-restimulated cultured splenocytes collected from allergic mice were also reduced by the treatment. The induction of interferon-γ and interleukin-4 secretion by splenocytes from ovalbumin-immunized mice stimulated in vitro with ovalbumin was also significantly reduced by the antigen complexed with anti-ovalbumin IgA. CONCLUSION: These data suggest that the direct inhibition of Th1 and Th2 activation by anti-ovalbumin monoclonal IgA participates in the inhibition of the primary antibody responses. IgA plays important immunosuppressive roles under physiological and pathological conditions and is a promising candidate drug for the treatment of immune disorders.

Ag and IL-2 immune complexes efficiently expand Ag-specific Treg cells that migrate in response to chemokines and reduce localized immune responses.

An intravenous administration of a high-dose antigen (Ag) can induce immune tolerance and suppress the immune response, but the mechanism remains unclear. We recently proved that a combined i.v. administration of OVA and IL-2-anti-IL-2 Ab immune complexes (IL-2 ICs) efficiently expands OVA-specific Treg cells in the thymus and induces their migration into peripheral blood, by using OVA-specific TCR Tg-expressing DO11.10 mice. Here, we demonstrate that the expanded OVA-specific Treg cells rapidly move into the air pouch after OVA injection in DO11.10 mice. The migration was inhibited by blocking the axis of a chemokine receptor, CCR2. Moreover, prior treatment with OVA and IL-2 ICs enhanced OVA-specific Treg-cell migration and inhibited OVA-induced delayed-type hypersensitivity (DTH) reactions in the skin of BM chimeric mice with 15% of T cells expressing OVA-specific TCR. Blocking the CCR2 axis reversed this suppression of DTH in these mice. Furthermore, prior treatment with OVA and IL-2 ICs effectively reduced DTH reactions even in WT mice possessing only a very small population of OVA-specific T cells. Thus, the treatment with Ag and IL-2 ICs can efficiently expand Ag-specific Treg cells with the capacity to migrate and reduce localized immune responses.

Elicitation of broadly reactive antibodies against glycan-modulated neutralizing V3 epitopes of HIV-1 by immune complex vaccines.

HIV-1 envelope gp120 is the target for neutralizing antibodies (NAbs) against the virus. Various approaches have been explored to improve immunogenicity of broadly neutralizing epitopes on this antigen with limited success. We previously demonstrated that immunogenicity of gp120 and especially its V3 epitopes was enhanced when gp120 was co-administered as immune-complex vaccines with monoclonal antibodies (mAb) to the CD4-binding site (CD4bs). To define the mechanisms by which immune complexes influence V3 immunogenicity, we compared gp120 complexed with mAbs specific for the C2 region (1006-30), the V2 loop (2158), or the CD4bs (654), and found that the gp120/654 and gp120/2158 complexes elicited anti-V3 NAbs, but the gp120/654 complex was the most effective. gp120 complexed with 654 F(ab')2 was as potent, indicating that V3 immunogenicity is determined by the specificity of the mAb's Fab fragment used to form the complexes. Importantly, the gp120/654 complex not only induced anti-gp120 antibodies (Abs) to higher titers, but also of greater avidity. The Abs were cross-reactive with V3 peptides from most subtype B and some subtype C isolates. Neutralization was detected only against Tier-1 HIV-1 pseudoviruses, while Tier-2 viruses, including the homologous JRFL strain, were not neutralized. However, JRFL produced in the presence of a mannosidase inhibitor was sensitive to anti-V3 NAbs in the immune sera. These results demonstrate that the gp120/654 complex is a potent immunogen for eliciting cross-reactive functional NAbs against V3 epitopes, of which exposure is determined by the specific compositions of glycans shrouding the HIV-1 envelope glycoproteins.

Results of a phase III clinical trial with an HBsAg-HBIG immunogenic complex therapeutic vaccine for chronic hepatitis B patients: experiences and findings.

BACKGROUND & AIMS: Even though various experimental therapeutic approaches for chronic hepatitis B infection have been reported, few of them have been verified by clinical trials. We have developed an antigen-antibody (HBsAg-HBIG) immunogenic complex therapeutic vaccine candidate with alum as adjuvant (YIC), aimed at breaking immune tolerance to HBV by modulating viral antigen processing and presentation. A double-blind, placebo-controlled, phase II B clinical trial of YIC has been reported previously, and herein we present the results of the phase III clinical trial of 450 patients. METHODS: Twelve doses of either YIC or alum alone as placebo were administered randomly to 450 CHB patients and they were followed for 24weeks after the completion of immunization. The primary end point was HBeAg seroconversion, and the secondary end points were decrease in viral load, improvement of liver function, and histology. RESULTS: In contrast to the previous phase II B trial using six doses of YIC and alum as placebo, six more injections of YIC or alum resulted in a decrease of the HBeAg seroconversion rate from 21.8% to 14.0% in the YIC group, but an increase from 9% to 21.9% in the alum group. Decrease in serum HBV DNA and normalization of liver function were similar in both groups (p>0.05). CONCLUSIONS: Overstimulation with YIC did not increase but decreased its efficacy due to immune fatigue in hosts. An appropriate immunization protocol should be explored and is crucial for therapeutic vaccination. Multiple injections of alum alone could have stimulated potent inflammatory and innate immune responses contributing to its therapeutic efficacy, and needs further investigation.

Mechanism of action and in vivo efficacy of a human-derived antibody against Staphylococcus aureus α-hemolysin.

The emergence and spread of multi-drug-resistant strains of Staphylococcus aureus in hospitals and in the community emphasize the urgency for the development of novel therapeutic interventions. Our approach was to evaluate the potential of harnessing the human immune system to guide the development of novel therapeutics. We explored the role of preexisting antibodies against S. aureus α-hemolysin in the serum of human individuals by isolating and characterizing one antibody with a remarkably high affinity to α-hemolysin. The antibody provided protection in S. aureus pneumonia, skin, and bacteremia mouse models of infection and also showed therapeutic efficacy when dosed up to 18 h post-infection in the pneumonia model. Additionally, in pneumonia and bacteremia animal models, the therapeutic efficacy of the α-hemolysin antibody appeared additive to the antibiotic linezolid. To better understand the mechanism of action of this isolated antibody, we solved the crystal structure of the α-hemolysin:antibody complex. To our knowledge, this is the first report of the crystal structure of the α-hemolysin monomer. The structure of the complex shows that the antibody binds α-hemolysin between the cap and the rim domains. In combination with biochemical data, the structure suggests that the antibody neutralizes the activity of the toxin by preventing binding to the plasma membrane of susceptible host cells. The data presented here suggest that protective antibodies directed against S. aureus molecules exist in some individuals and that such antibodies have a therapeutic potential either alone or in combination with antibiotics.

Immunological complex for enhancement of innate immune response in passive vaccination.

Passive vaccination is used to treat a wide range of infections and cancer. However, this approach has some limitations. An immune complex termed Y-complex was developed to intensify the effect of the passive vaccine. The complex is composed of a microbead that carries specific antibodies and an inducer. It enables targeting of pathogen or abnormal cells, and stimulation of a desired response by innate immune cells, depending on the inducer. The production and efficacy of Y-complex as a passive immune prophylaxis is demonstrated in this study by its use in treating cow mastitis. In an in vitro assay, Y-complex inhibited propagation and induced phagocytosis of bacteria. In challenge experiments, cows were inoculated through the udder with Escherichia coli or Streptococcus dysgalactiae. Following treatment with Y-complex, no bacteria were isolated in the milk and N-acetyl-ß-D-glucosaminidase activity had returned to normal levels. Thus the Y-complex approach can be used as an effective treatment for mastitis. Due to its modularity, this approach may serve as a treatment for a variety of disease agents.

Noncovalent assembly of anti-dendritic cell antibodies and antigens for evoking immune responses in vitro and in vivo.

Targeting of Ags directly to dendritic cells (DCs) through anti-DC receptor Ab fused to Ag proteins is a promising approach to vaccine development. However, not all Ags can be expressed as a rAb directly fused to a protein Ag. In this study, we show that noncovalent assembly of Ab-Ag complexes, mediated by interaction between dockerin and cohesin domains from cellulose-degrading bacteria, can greatly expand the range of Ags for this DC-targeting vaccine technology. rAbs with a dockerin domain fused to the rAb H chain C terminus are efficiently secreted by mammalian cells, and many Ags not secreted as rAb fusion proteins are readily expressed as cohesin directly fused to Ag either via secretion from mammalian cells or as soluble cytoplasmic Escherichia coli products. These form very stable and homogeneous complexes with rAb fused to dockerin. In vitro, these complexes can efficiently bind to human DC receptors followed by presentation to Ag-specific CD4⁺ and CD8⁺ T cells. Low doses of the HA1 subunit of influenza hemagglutinin conjugated through this means to anti-Langerin rAbs elicited Flu HA1-specific Ab and T cell responses in mice. Thus, the noncovalent assembly of rAb and Ag through dockerin and cohesin interaction provides a useful modular strategy for development and testing of prototype vaccines for elicitation of Ag-specific T and B cell responses, particularly when direct rAb fusions to Ag cannot be expressed.

Critical role for CCAAT/enhancer-binding protein ß in immune complex-induced acute lung injury.

C/EBPs, particularly C/EBPß and C/EBPδ, are known to participate in the regulation of many genes associated with inflammation. However, very little is known regarding the activation and functions of C/EBPß and C/EBPδ in acute lung inflammation and injury. In this study, we show that both C/EBPß and C/EBPδ activation are triggered in lungs and in alveolar macrophages following intrapulmonary deposition of IgG immune complexes. We further show that mice carrying a targeted deletion of the C/EBPß gene displayed significant attenuation of the permeability index (lung vascular leak of albumin), lung neutrophil accumulation (myeloperoxidase activity), total number of WBCs, and neutrophils in bronchoalveolar lavage fluids compared with wild-type mice. Moreover, the mutant mice expressed considerably less TNF-α, IL-6, and CXC/CC chemokine and soluble ICAM-1 proteins in bronchoalveolar lavage fluids, and corresponding mRNAs in the IgG immune complex-injured lung, compared with wild-type mice. These phenotypes were associated with a significant reduction in morphological lung injury. In contrast, C/EBPδ deficiency had no effect on IgG immune complex-induced lung injury. IgG immune complex-stimulated C/EBPß-deficient alveolar macrophages released significantly less TNF-α, IL-6, MIP-2, keratinocyte cell-derived chemokine, and MIP-1α compared with wild-type cells. Similar decreases in IgG immune complex-induced inflammatory mediator production were observed following small interfering RNA ablation of C/EBPß in a murine alveolar macrophage cell line. These findings implicate C/EBPß as a critical regulator of IgG immune complex-induced inflammatory responses and injury in the lung.

Regulatory T cells expressing granzyme B play a critical role in controlling lung inflammation during acute viral infection.

The inflammatory response to lung infections must be tightly regulated, enabling pathogen elimination while maintaining crucial gas exchange. Using recently described "depletion of regulatory T cell" (DEREG) mice, we found that selective depletion of regulatory T cells (Tregs) during acute respiratory syncytial virus (RSV) infection enhanced viral clearance but increased weight loss, local cytokine and chemokine release, and T-cell activation and cellular influx into the lungs. Conversely, inflammation was decreased when Treg numbers and activity were boosted using interleukin-2 immune complexes. Unexpectedly, lung (but not draining lymph node) Tregs from RSV-infected mice expressed granzyme B (GzmB), and bone marrow chimeric mice with selective loss of GzmB in the Treg compartment displayed markedly enhanced cellular infiltration into the lung after infection. A crucial role for GzmB-expressing Tregs has not hitherto been described in the lung or during acute infections, but may explain the inability of children with perforin/GzmB defects to regulate immune responses to infection. The effects of RSV infection in mice with defective immune regulation closely parallel the observed effects of RSV in children with bronchiolitis, suggesting that the pathogenesis of bronchiolitis may involve an inability to regulate virus-induced inflammation.

Establishment and characterization of a murine model for allergic asthma using allergen-specific IgE monoclonal antibody to study pathological roles of IgE.

Allergen-specific IgE has long been regarded as a major molecular component of allergic asthma. Although IgE plays a central role in the early asthmatic response, its roles in the chronic phase, such as the late asthmatic response, airway hyperresponsiveness (AHR), and airway remodeling (goblet cell hyperplasia and subepithelial fibrosis) have not yet been defined well. In this study, we investigated the hypothesis that chronic responses could be induced by IgE-dependent mechanisms. BALB/c mice passively sensitized with an ovalbumin (OVA)-specific IgE monoclonal antibody (mAb) were repeatedly challenged with intratracheal administration of OVA. The first challenge induced early phase airway narrowing without any late response, but the fourth challenge caused not only an early but also a late phase response, AHR, and goblet cell hyperplasia. Macrophages, lymphocytes and neutrophils, but not eosinophils, were significantly increased in the lung 24h after the fourth challenge. Interestingly, levels of OVA-specific IgG1 in serum increased by multiple antigen challenges. A C3a receptor antagonist inhibited the late asthmatic response, AHR, and infiltration by neutrophils. In contrast, no late response, goblet cell hyperplasia, inflammatory cells, or production of IgG1 was observed in severe combined immunodeficient mice. On the other hand, seven challenges in BALB/c mice induced subepithelial fibrosis associated with infiltration by eosinophils. In conclusion, the allergic asthmatic responses induced by passive sensitization with IgE mAb can provide a useful model system to study the pathological roles of IgE in acute and chronic phases of allergic asthma.

Production of heterologous IgG antibody against Heymann nephritis antigen by injections of immune complexes.

Heterologous IgG antibody (ab) can be produced against Heymann nephritis (HN) antigen (ag) in rabbits by administering it in Freund's complete adjuvant. The developing abs reacted at high titre with rat kidney brush border (BB) regions of the renal proximal tubules in an indirect fluorescence ab test. A single IV injection of the heterologous ab into a susceptible strain of rat resulted in the localization of IgG ab to glomerular fixed ags, producing immune complex glomerular nephritis. The injected ab also reacted with the BB region of the renal proximal tubules. The aim of this experiment was to find out whether heterologous IgG ab against the HN ag can also be produced in recipient rabbits by injecting immune complexes (ICs) composed of a rat kidney tubular preparation [rat kidney fraction 3 (rKF3)] and donor rabbit-derived rabbit anti-rKF3 IgG ab. We found that anti-rKF3 IgG ab--against the BB region of the renal proximal tubules--could be induced in rabbits injected with ICs, and the resulting ab was able to initiate passive HN in rats. This was the first time a pathogenic IgG ab was produced against HN ag in rabbits without the use of adjuvant. Ab responses in recipient rabbits were achieved by ab information transfer. Recipient rabbits injected with the IC produced the same class of immunoglobulin with the same specificity against the target ag rKF3, as was present in the innoculum, namely rabbit anti-rKF3 IgG ab.

Recombinant glycoprotein B vaccine formulation with Toll-like receptor 9 agonist and immune-stimulating complex induces specific immunity against multiple strains of cytomegalovirus.

Natural human cytomegalovirus (CMV) infection is characterized by a strain-specific neutralizing antibody response. This is particularly relevant in clinical settings such as transplantation and pregnancy where reinfection with heterologous strains occurs and the immune system does not mount an effective response against the infecting strain due to underlying immunosuppression. There is an emerging argument that a CMV vaccine that induces high titres of cross-neutralizing antibodies will be more effective in protecting individuals from infection with antigenically different CMV strains. In addition, induction of cell-mediated immunity offers the additional advantage of targeting virus-infected cells. This study presents a novel formulation of a CMV vaccine that, by combining recombinant soluble gB protein with a Toll-like receptor 9 agonist (CpG ODN1826) and immune-stimulating complexes (AbISCO 100), was able to elicit strong polyfunctional CMV-specific cellular and cross-neutralizing humoral immune responses. These data demonstrated that prime-boost immunization of human leukocyte antigen (HLA)-A2 mice with gB protein in combination with CpG ODN1826 and AbISCO 100 induced long-lasting CMV-specific CD4(+) and CD8(+) T-cell and humoral responses. Furthermore, these responses neutralized infection with multiple strains of CMV expressing different gB genotypes and afforded protection against challenge with recombinant vaccinia virus encoding the gB protein. These observations argue that this novel vaccine strategy, if applied to humans, should facilitate the generation of a robust, pluripotent immune response, which may be more effective in preventing infection with multiple strains of CMV.

Effect of an immunogenic complex containing WHV viral particles and non-neutralizing anti-HBs antibodies on the outcome of WHV infection in woodchucks.

The Eastern woodchuck (Marmota monax) is a useful experimental model for evaluating antiviral therapy against chronic HBV infection. In the present study, an immunogenic complex (IGC) composed of immune sera containing PreS/S heterologous antibodies (anti-HBs) and serum-derived WHV particles containing 10(7) WHV-DNA copies/50 µl was developed. The IGC was administered to WHV-negative woodchucks and natural chronic WHV carriers, with the final aim of evaluating the outcome of WHV infection in both groups. A control group of three animals, infected experimentally with viral particles only, was also evaluated. Following IGC administration, two WHV-negative woodchucks exhibited persistent infection, with WHV-DNA levels 3-6 logs lower than the WHV-DNA levels of the controls that developed persistent infection. WHeAg seroconversion to anti-WHe was observed in these two woodchucks and in two control woodchucks which developed self-limited infection. In two of the four chronic carriers, the WHV-DNA level decreased significantly (by 4-6 logs) following IGC administration, with no rebound in viral load during follow-up. WHeAg seroconversion to anti-WHe was observed also in these animals. Analyses of the sequences derived from envelope proteins confirmed that IGC did not induce the emergence of resistant viral variants. The results of this study indicate that the IGC could be useful for breaking the tolerance in hepadnaviral infection and for boosting the host's innate and adoptive immune response.