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1.  IgG3 deficiency extends lifespan and attenuates progression of glomerulonephritis in MRL/lpr mice 
Biology Direct  2012;7:3.
Antibodies of the IgG3 subclass have been implicated in the pathogenesis of the spontaneous glomerulonephritis observed in mice of the MRL/MpJ-Tnfrsf6lpr (MRL/lpr) inbred strain which have been widely studied as a model of systemic lupus erythematosus We have produced IgG3-deficient (-/-) mice with the MRL/lpr genetic background to determine whether IgG3 antibodies are necessary for or at least contributory to MRL/lpr-associated nephritis.
The gamma3 genotype (+/+ vs. +/- vs. -/-) did not appear to significantly affect serum titers of IgG auto-antibodies specific for double-stranded DNA (dsDNA) or α-actinin. However, while substantial serum titers of IgG3 auto-antibodies specific for double-stranded DNA (dsDNA) or α-actinin were seen in gamma3 +/+ mice, somewhat lower serum titers of these IgG3 auto-antibodies were found in gamma3 +/- mice, and gamma3 -/- mice exhibited baseline concentrations of these auto-antibodies. Analysis of immunoglobulins eluted from snap-frozen kidneys obtained from mice of all three gamma3 genotypes at ~18 weeks of age revealed much higher quantities of IgG in the kidneys from gamma3 +/+ than gamma3 -/- mice, and most IgG eluted from +/+ mice was IgG3. The serum creatinine levels in gamma3 +/+ mice substantially exceeded those of age-matched gamma3 -/- mice after ~21 weeks of age. Histopathological examination of kidneys from mice sacrificed at pre-determined ages also revealed more extensive glomerulosclerosis in gamma3 +/+ or +/- mice than in -/- mice beginning at 21 weeks of age. Survival analysis for IgG3-deficient and IgG3-producing MRL/lpr mice revealed that gamma3 -/- mice lived significantly longer (p = 0.0006) than either gamma3 +/- or +/+ mice. Spontaneous death appeared to be due to irreversible renal failure, because > 85% of glomeruli in kidneys from mice that died spontaneously were obliterated by glomerulosclerosis.
The available evidence suggests that IgG3 deficiency partially protects MRL/lpr mice against glomerulonephritis-associated morbidity and mortality by slowing or arresting the progression to glomerulosclerosis.
This article was reviewed by Pushpa Pandiyan, Irun Cohen, and Etienne Joly.
PMCID: PMC3293080  PMID: 22248284
2.  Outer Membrane Protein Complex of Meningococcus Enhances the Antipolysaccharide Antibody Response to Pneumococcal Polysaccharide–CRM197 Conjugate Vaccine ▿ 
Bacterial polysaccharides (PS) are T cell-independent antigens that do not induce immunologic memory and are poor immunogens in infants. Conjugate vaccines in which the PS is covalently linked to a carrier protein have enhanced immunogenicity that resembles that of T cell-dependent antigens. The Haemophilus influenzae type b (Hib) conjugate vaccine, which uses the outer membrane protein complex (OMPC) from meningococcus as a carrier protein, elicits protective levels of anti-capsular PS antibody (Ab) after a single dose, in contrast to other conjugate vaccines, which require multiple doses. We have previously shown that OMPC robustly engages Toll-like receptor 2 (TLR2) and enhances the early anti-Hib PS Ab titer associated with an increase in TLR2-mediated induction of cytokines. We now show that the addition of OMPC to the 7-valent pneumococcal PS-CRM197 conjugate vaccine during immunization significantly increases the anti-PS IgG and IgM responses to most serotypes of pneumococcus contained in the vaccine. The addition of OMPC also increased the likelihood of anti-PS IgG3 production against serotypes 4, 6B, 9V, 18C, 19F, and 23F. Splenocytes from mice who had received OMPC with the pneumococcal conjugate vaccine produced significantly more interleukin-2 (IL-2), IL-4, IL-6, IL-10, tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) than splenocytes from mice who received phosphate-buffered saline (PBS) plus the conjugate vaccine. We conclude that OMPC enhances the anti-PS Ab response to pneumococcal PS-CRM197 conjugate vaccine, an effect associated with a distinct change in cytokine profile. It may be possible to reduce the number of conjugate vaccine doses required to achieve protective Ab levels by priming with adjuvants that are TLR2 ligands.
PMCID: PMC3122513  PMID: 21450979
3.  CpG Oligodeoxynucleotides Act as Adjuvants for Pneumococcal Polysaccharide-Protein Conjugate Vaccines and Enhance Antipolysaccharide Immunoglobulin G2a (IgG2a) and IgG3 Antibodies 
Infection and Immunity  2000;68(3):1450-1456.
Pneumococcal polysaccharide-protein conjugate vaccines elicit antipolysaccharide antibodies, but multiple doses are required to achieve protective antibody levels in children. In addition, the immunogenicity of experimental multivalent pneumococcal conjugate vaccines varies with different polysaccharide serotypes. One strategy to improve these vaccines is to incorporate an adjuvant to enhance their immunogenicity. Synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) are adjuvants that promote T-cell and T-dependent antibody responses to protein antigens, but it has been unclear whether CpG ODN can enhance polysaccharide-specific antibody responses. The present studies demonstrate significant adjuvant activity of CpG ODN for antibody responses against Streptococcus pneumoniae polysaccharide types 19F and 6B induced by conjugates of 19F and 6B with the protein carrier CRM197. BALB/c ByJ mice were injected with 19F-CRM197 or 6B-CRM197 with or without CpG ODN, and sera were tested for anti-19F or anti-6B antibodies by enzyme-linked immunosorbent assay. The polysaccharide-specific antibody response to 19F-CRM197 alone was predominantly of the immunoglobulin G1 (IgG1) and IgM isotypes, but addition of CpG ODN markedly increased geometric mean titers of total anti-19F antibody (23-fold), anti-19F IgG2a (26-fold), and anti-19F IgG3 (>246-fold). The polysaccharide-specific antibody response to 6B-CRM197 alone consisted only of IgM, but addition of CpG ODN induced high titers of anti-6B IgG1 (>78-fold increase), anti-6B IgG2a (>54-fold increase), and anti-6B IgG3 (>3,162-fold increase). CpG ODN also increased anti-CRM197 IgG2a and IgG3. Adjuvant effects were not observed with control non-CpG ODN. Thus, CpG ODN significantly enhance antipolysaccharide IgG responses (especially IgG2a and IgG3) induced by these glycoconjugate vaccines.
PMCID: PMC97300  PMID: 10678959
4.  B- and T-Cell Immune Responses to Pneumococcal Conjugate Vaccines: Divergence between Carrier- and Polysaccharide-Specific Immunogenicity 
Infection and Immunity  1999;67(9):4862-4869.
Conjugation of various serotypes of pneumococcal polysaccharide (PnPS) to carrier protein enhances the magnitude of the polysaccharide-specific antibody response, presumably by eliciting T-cell help. However, variability in PnPS serotype-specific immunogenicity has been observed. CBA/J mice immunized with either 6B or 19F PnPS conjugated to the protein carrier Cross Reactive Material197 (CRM197) produce a strong anti-PnPS antibody response; however, when mice are immunized with 23F PnPS conjugated to CRM197, they fail to produce a significant anti-PnPS response. In order to determine whether this difference was related to alterations in antigen processing of the carrier protein and the subsequent T-cell responses, we studied proliferation of lymphocytes from CBA/J mice immunized with CRM197 alone or conjugated to 6B, 19F, or 23F PnPS. T-cell proliferative responses to synthetic peptides demonstrated that lymph node cells elicited by the poorly immunogenic conjugate 23F-CRM197 recognized many, but not all, of the epitopes recognized by lymph node cells elicited by 6B- and 19F-CRM197 as well as additional epitopes. Despite marked differences in PnPS-specific immunogenicity, all mice made high titers of CRM197 antibodies of the immunoglobulin G1 isotype. Cells from mice immunized with any of the conjugates yielded vigorous T-cell responses to whole antigen. We conclude that the serotype of PnPS can alter the peptide specificities of T-cell responses, but even a poorly immunogenic PnPS conjugate can elicit a significant T-cell response. Thus, conjugation of PnPS to a carrier protein that elicits carrier-specific T- and B-cell responses does not necessarily enhance PnPS immunogenicity.
PMCID: PMC96820  PMID: 10456942
5.  A Human Anti-Pseudomonas aeruginosa Serotype O6ad Immunoglobulin G1 Expressed in Transgenic Tobacco Is Capable of Recruiting Immune System Effector Function In Vitro▿  
The production of a recombinant human IgG1 in transgenic tobacco was examined to determine whether a plant-derived antibody could recruit immune system effector function against a bacterial pathogen. A plant transformation vector was engineered to contain genes for a human kappa light chain and a human gamma-1 heavy chain with VH and VL sequences from a previously identified human IgG2 monoclonal antibody (MAb) that specifically binds to and opsonizes Pseudomonas aeruginosa serotype O6ad. Unique NcoI and NotI restriction sites were incorporated to flank these variable sequences, resulting in a plant transformation vector that could be engineered for expression of any other human IgG1 antibody, requiring only the substitution of other VH and VL antigen-binding coding sequences. The plant-produced IgG1 was determined to have high-mannose glycan content and to be capable of mediating opsonophagocytosis of P. aeruginosa serotype O6ad in vitro using human complement and human polymorphonuclear leukocytes. Thus, MAbs produced in plants from this vector could provide human IgG1 MAbs for targeting other pathogens that require the recruitment of immune system effector functions.
PMCID: PMC2043195  PMID: 17606688
6.  Clearance of Citrobacter rodentium Requires B Cells but Not Secretory Immunoglobulin A (IgA) or IgM Antibodies  
Infection and Immunity  2004;72(6):3315-3324.
Citrobacter rodentium, a murine model pathogen for human enteropathogenic Escherichia coli, predominantly colonizes the lumen and mucosal surface of the colon and cecum and causes crypt hyperplasia and mucosal inflammation. Mice infected with C. rodentium develop a secretory immunoglobulin A (IgA) response, but the role of B cells or secretory antibodies in host defense is unknown. To address this question, we conducted oral C. rodentium infections in mice lacking B cells, IgA, secreted IgM, polymeric Ig receptor (pIgR), or J chain. Normal mice showed peak bacterial numbers in colon and feces at 1 week and bacterial eradication after 3 to 4 weeks. B-cell-deficient mice were equally susceptible initially but could not control infection subsequently. Tissue responses showed marked differences, as infection of normal mice was accompanied by transient crypt hyperplasia and mucosal inflammation in the colon and cecum at 2 but not 6 weeks, whereas B-cell-deficient mice had few mucosal changes at 2 weeks but severe epithelial hyperplasia with ulcerations and mucosal inflammation at 6 weeks. The functions of B cells were not mediated by secretory antibodies, since mice lacking IgA or secreted IgM or proteins required for their transport into the lumen, pIgR or J chain, cleared C. rodentium normally. Nonetheless, systemic administration of immune sera reduced bacterial numbers significantly in normal and pIgR-deficient mice, and depletion of IgG abrogated this effect. These results indicate that host defense against C. rodentium depends on B cells and IgG antibodies but does not require production or transepithelial transport of IgA or secreted IgM.
PMCID: PMC415672  PMID: 15155635
7.  Antigen Processing of the Heptavalent Pneumococcal Conjugate Vaccine Carrier Protein CRM197 Differs Depending on the Serotype of the Attached Polysaccharide  
Infection and Immunity  2003;71(7):4186-4189.
The pneumococcal (Pn) conjugate vaccine includes seven different polysaccharides (PS) conjugated to CRM197. Utilizing antigen-processing cells and a CRM197-specific mouse T-cell hybridoma, we found that the serotype of conjugated PnPS dramatically affected antigen processing of CRM197. Unconjugated CRM197 and serotype conjugates 14 and 18C were processed more efficiently.
PMCID: PMC161992  PMID: 12819115
8.  Enhanced Immunogenicity of Pneumococcal Surface Adhesin A by Genetic Fusion to Cytokines and Evaluation of Protective Immunity in Mice  
Infection and Immunity  2002;70(10):5589-5595.
Immunization of mice with pneumococcal surface adhesin A (PsaA) emulsified in complete Freund's adjuvant (CFA) provides protection against systemic infection with Streptococcus pneumoniae. Because the use of CFA is not acceptable in humans, we sought to develop alternative means of enhancing the immunogenicity of protein antigens of potential use in pneumococcal vaccines. We designed a series of genetic constructs in which coding sequences for PsaA were linked to sequences encoding either murine interleukin-2 (mIL-2), mIL-4, or two copies of an immunostimulatory nonapeptide derived from mIL-1β. The PsaA-cytokine constructs were cloned and expressed in Escherichia coli. Mice immunized twice with PsaA-IL-2, or PsaA-IL-4 responded with PsaA-specific antibody production comparable in magnitude to that of mice primed with PsaA in CFA and boosted with PsaA in incomplete Freund's adjuvant (PsaA-Adj). Antibodies elicited by PsaA-Adj were predominantly of the immunoglobulin G1 (IgG1) subclass, while PsaA-IL-2 and PsaA-IL-4 elicited substantial amounts of IgG2a in addition to IgG1. Mice immunized with PsaA-Adj or PsaA-IL-4 were partially protected against intraperitoneal challenge with virulent S. pneumoniae (30% overall survival beyond 15 days postchallenge). Mice immunized with PsaA and no adjuvant or PsaA-IL-2 exhibited 0 or 5% survival rates, respectively, following challenge. In contrast, mice immunized twice with capsular polysaccharide were 100% protected. The modest levels of protection seen in mice immunized with PsaA and its more immunogenic derivatives may be explained in part by the relative inaccessibility of antibody to PsaA on the surface of encapsulated S. pneumoniae.
PMCID: PMC128336  PMID: 12228286
9.  Human Monoclonal Antibodies against Pseudomonas aeruginosa Lipopolysaccharide Derived from Transgenic Mice Containing Megabase Human Immunoglobulin Loci Are Opsonic and Protective against Fatal Pseudomonas Sepsis 
Infection and Immunity  2001;69(4):2223-2229.
Pseudomonas aeruginosa is a significant human pathogen, and no vaccine is commercially available. Passive antibody prophylaxis using monoclonal antibodies (MAb) against protective P. aeruginosa epitopes is an alternative strategy for preventing P. aeruginosa infection, but mouse MAb are not suitable for use in humans. Polyclonal human antibodies from multiple donors have variable antibody titers, and human MAb are difficult to make. We used immunoglobulin-inactivated transgenic mice reconstituted with megabase-size human immunoglobulin loci to generate a human MAb against the polysaccharide (PS) portion of the lipopolysaccharide O side chain of a common pathogenic serogroup of P. aeruginosa, 06ad. The anti-PS human immunoglobulin G2 MAb made from mice immunized with heat-killed P. aeruginosa was specific for serogroup 06ad pseudomonas. The MAb was highly opsonic for the uptake and killing of P. aeruginosa by human polymorphonuclear leukocytes in the presence of human complement. In addition, 25 μg of the MAb protected 100% of neutropenic mice from fatal P. aeruginosa sepsis. DNA sequence analysis of the genes encoding the MAb revealed VH3 and Vκ2/A2 variable-region genes, similar to variable-region genes in humans immunized with bacterial PS and associated with high-avidity anti-PS antibodies. We conclude that human MAb to P. aeruginosa made in these transgenic mice are highly protective and that these mice mimic the antibody response seen in humans immunized with T-cell-independent antigens such as bacterial PS.
PMCID: PMC98149  PMID: 11254577
10.  Efficacy of Human Hyperimmune Globulin in Prevention of Haemophilus influenzae Type b Disease in Infant Rats 
Infection and Immunity  1983;39(2):709-714.
To determine the protective efficacy of human hyperimmune globulin to Haemophilus influenzae type b disease in an infant rat model, we compared hyperimmune globulin containing 600 μg of anti-polyribophosphate (PRP) antibody per ml to conventional immune globulin containing 66 μg of anti-PRP antibody per ml. The hyperimmune globulin was fractionated from the pooled plasma of 55 adult donors immunized with PRP, the capsular polysaccharide of H. influenzae type b. The disappearance of passively administered antibody was biphasic, with a linear first-order disappearance curve during the first 7 days. The initial half-life for anti-PRP antibody was 2.38 days in rats nasally colonized but not detectably bacteremic with H. influenzae type b and significantly longer (half-life, 10.3 days; P < 0.01) in noncolonized animals. Hyperimmune globulin afforded 10 times the protection of conventional globulin against bacteremia and meningitis. Globulin depleted of anti-PRP antibody offered no protection. The initial serum antibody levels and the levels during the 8-day observation period predicted protection. Rats maintaining serum antibody levels greater or equal to 50 ng/ml to day 8 had a 10% bacteremia and 5% meningitis incidence in contrast with 95% bacteremia (P < 0.001) and 55% meningitis (P < 0.001) in rats with less than 50 ng of anti-PRP antibody per ml. We conclude that studies of the pharmacology and efficacy of hyperimmune globulin are warranted in high-risk children unable to respond to active immunization.
PMCID: PMC348007  PMID: 6601060

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