Class Switch Recombination Defects: impact on B cell maturation and antibody responses.

To assess how B cell phenotype analysis correlates with antigen responses in patients with class switch recombination defects (CSRD) we quantified memory B cells by flow-cytometry and immunized CSRD patients with the neoantigen bacteriophage phiX174 (phage). CSRD patients showed uniformly absent or markedly reduced switched memory B cells (IgM-IgD-CD27+). CD40L patients had reduced CD27+ memory B cells (both non-switched and switched). In NEMO patients, results varied depending on the IKKγ gene variant. Three of four AID patients had normal percentages of CD27+ memory B cells while CD27+IgM-IgD- switched memory B cells were markedly reduced in all AID patients. Antibody response to phage was remarkably decreased with lack of memory amplification and class-switching in immunized CD40L, UNG deficient, and NEMO patients. Distinct B-cell phenotype pattern correlated with abnormal antibody responses to a T-cell dependent neoantigen, representing a powerful tool to identify CSRD patients.

Production and characterization of mouse monoclonal antibodies against lysis protein E of phiX174.

Bacterial ghosts offer a new avenue for the study of inactivated vaccines. However, for many years the mechanism of genetic inactivation was controversial. To obtain mouse monoclonal antibodies (mAbs) against protein E will allow the observation of its location and dynamic expression to expand understanding of the lysis mechanism. In this study, a His-tagged ΔE fusion protein expressed in bacteria was used as the immunogen, and mAbs targeting protein E were produced by the hybridoma technique and selected by enzyme-linked immunosorbent assay using GST-E and GST-ΔE as coating proteins. Purified mAbs from mouse ascites were screened against a phage-displayed random dodecapeptide library (Ph.D.-12). After three rounds of biopanning, 30 phage clones were randomly selected and sequenced, and their amino acids were deduced. One epitope showed a good match with protein E at 57-63aa (KPLN--R) and the synthetic decapeptide Epep (VRLKPLNCSR) strongly inhibited combination of E-A5 with E fusion proteins. Immunofluorescence microscopy indicated specific immunoreactivity of E-A5 with Escherichia coli-expressed native protein E. The novel mAbs may be of great potential value in analysis of the function and lysis pathway of protein E and other relative phages and in evaluation of E as potential marker of bacterial ghosts.

Use and interpretation of diagnostic vaccination in primary immunodeficiency: a working group report of the Basic and Clinical Immunology Interest Section of the American Academy of Allergy, Asthma & Immunology.

A major diagnostic intervention in the consideration of many patients suspected to have primary immunodeficiency diseases (PIDDs) is the application and interpretation of vaccination. Specifically, the antibody response to antigenic challenge with vaccines can provide substantive insight into the status of human immune function. There are numerous vaccines that are commonly used in healthy individuals, as well as others that are available for specialized applications. Both can potentially be used to facilitate consideration of PIDD. However, the application of vaccines and interpretation of antibody responses in this context are complex. These rely on consideration of numerous existing specific studies, interpolation of data from healthy populations, current diagnostic guidelines, and expert subspecialist practice. This document represents an attempt of a working group of the American Academy of Allergy, Asthma & Immunology to provide further guidance and synthesis in this use of vaccination for diagnostic purposes in consideration of PIDD, as well as to identify key areas for further research.

Immune responses in adult female volunteers during the bed-rest model of spaceflight: antibodies and cytokines.

BACKGROUND: It is unknown whether a prolonged period of bed rest will affect human immune responses, particularly in female subjects. OBJECTIVE: We sought to measure immune responses in adult female subjects exposed to prolonged bed rest. METHODS: Adult (25-40 years) female volunteers (n = 24) were maintained in a supine (6 degrees tilt) head-down bed-rest (HDBR) position for 60 days: 8 with HDBR only, 8 with HDBR and regular muscular exercise, and 8 with HDBR and dietary protein supplementation. Subjects were immunized with bacteriophage phiX-174. Antibody production and plasma cytokine levels were determined. RESULTS: The rate of primary antibody production of the HDBR plus exercise group increased faster (P = .01) and to a higher level versus that of the HDBR-only group (P = .03) and that of the HDBR plus diet group (trend P = .08). The rates of secondary antibody production between the 3 groups were similar, but the level of antibody in the HDBR plus exercise group remained higher versus that in the HDBR-only group (P = .03). Both the HDBR (P = .001) and HDBR plus diet (P = .02) groups had time-related progressive increases in TNF-alpha receptor levels, but the HDBR plus exercise group remained at baseline. The HDBR plus exercise group experienced an acute increase in IL-1 receptor antagonist levels versus the HDBR (P = .02) and the HDBR plus diet (P = .02) groups, with similar increases in RANTES levels. CONCLUSIONS: The exercise countermeasure accelerated primary antibody production and increased antibody levels to bacteriophage phiX-174 and also opposed the potentially harmful effects of increased TNF-alpha levels caused by prolonged bed rest, possibly by activating the anti-inflammatory cytokine IL-1 receptor antagonist and the chemotactic factor RANTES.

Immunologic reconstitution during PEG-ADA therapy in an unusual mosaic ADA deficient patient.

We report detailed genetic and immunologic studies in a patient diagnosed with adenosine deaminase (ADA) deficiency and combined immune deficiency at age 5 years. At the time of diagnosis, although all other lymphocyte subsets were depleted, circulating CD8(+) T cells with a terminally differentiated phenotype were abundant and expressed normal ADA activity due to a reversion mutation in a CD8(+) T cell or precursor. Over the first 9 months of replacement therapy with PEG-ADA, the patient steadily accumulated mature naïve CD4(+) and CD8(+) T cells, as well as CD4(+)/FOXP3(+) regulatory T cells, consistent with restoration of a functional cellular immune system. While CD19(+) naïve B cells also accumulated in response to PEG-ADA therapy, a high proportion of these B cells exhibited an immature surface marker phenotype even after 9 months, and immunization with neoantigen bacteriophage varphiX174 demonstrated a markedly subnormal humoral immune response. Our observations in this single patient have important implications for gene therapy of human ADA deficiency, as they indicate that ADA expression within even a large circulating lymphocyte population may not be sufficient to support adequate immune reconstitution. They also suggest that an immature surface marker phenotype of the peripheral B cell compartment may be a useful surrogate marker for incomplete humoral immune reconstitution during enzyme replacement, and possibly other forms of hematopoietic cell therapies.

Balancing immunity and immunosuppression: vaccinating patients receiving treatment with efalizumab.

Although effective in the treatment of immunodysregulatory diseases such as psoriasis, targeted immunosuppressive agents may confer risks of both enhanced susceptibility to infection and decreased responsiveness to vaccination. In a recent study, Krueger et al. (this issue) investigated these issues by testing the immune response to both a model antigen and a therapeutic vaccination in psoriasis patients during and after treatment with an LFA-1 inhibitor, efalizumab.

Transient blockade of the inducible costimulator pathway generates long-term tolerance to factor VIII after nonviral gene transfer into hemophilia A mice.

Formation of inhibitory antibodies is a common problem encountered in clinical treatment for hemophilia. Human factor VIII (hFVIII) plasmid gene therapy in hemophilia A mice also leads to strong humoral responses. We demonstrate that short-term therapy with an anti-ICOS monoclonal antibody to transiently block the inducible costimulator/inducible costimulator ligand (ICOS/ICOSL) signaling pathway led to sustained tolerance to hFVIII in hFVIII plasmid-treated hemophilia A mice and allowed persistent, high-level FVIII functional activity (100%-300% of normal). Anti-ICOS treatment resulted in depletion of ICOS(+)CD4(+) T cells and activation of CD25(+)Foxp3(+) Tregs in the peripheral blood, spleen, and lymph nodes. CD4(+) T cells from anti-ICOS-treated mice did not proliferate in response to hFVIII stimulation and produced high levels of regulatory cytokines, including interleukin-10 and transforming growth factor-beta. Moreover, CD4(+)CD25(+) Tregs from tolerized mice adoptively transferred dominant tolerance in syngeneic hFVIII plasmid-treated hemophilia A mice and reduced the production of antibodies against FVIII. Anti-ICOS-treated mice tolerized to hFVIII generated normal primary and secondary antibody responses after immunization with the T-dependent antigen, bacteriophage Phix 174, indicating maintenance of immune competency. Our data indicate that transient anti-ICOS monoclonal antibody treatment represents a novel single-agent immunomodulatory strategy to overcome the immune responses against transgene product after gene therapy.

Effect of therapeutic integrin (CD11a) blockade with efalizumab on immune responses to model antigens in humans: results of a randomized, single blind study.

Efalizumab is a humanized monoclonal CD11a antibody approved for treatment of psoriasis. Its immunomodulatory effects led us study how immune responses are modified and the possible consequences for vaccinations in clinical practice. This was a randomized, single-blind, placebo-controlled, parallel-group study of 12 weeks of subcutaneous efalizumab treatment of patients with moderate psoriasis. Bacteriophage phiX174 was used as a model neoantigen to assess T-cell-dependent humoral immunity. Tetanus booster vaccine, pneumococcal vaccine, and intracutaneous skin tests were administered to further evaluate humoral and cellular immune responses. During efalizumab treatment, both primary and secondary antibody responses to phiX174, including IgM/IgG isotype switch, were reduced. There appeared to be naïve T-cell anergy to a neoantigen (phiX174) during active CD11a blockade, without tolerance to the antigen after efalizumab withdrawal. Secondary humoral immune responses to tetanus booster during treatment were reduced, but antibody titer increases led to protective levels. Responses to pneumococcal vaccination 6 weeks after withdrawal from efalizumab were not affected. Cellular immune responses to intracutaneous recall antigens were reduced during treatment and returned to pretreatment conditions after withdrawal. These results expand our knowledge of how immune responses are modulated in humans by CD11a blockade and have implications for vaccinations of patients treated with this agent.

A transgenic mouse model for studying the clearance of blood-borne pathogens via human complement receptor 1 (CR1).

Complement receptor 1 (CR1) on the surface of human erythrocytes facilitates intravascular clearance of complement-opsonized pathogens. The need for complement activation can be circumvented by directly coupling the organism to CR1 using a bispecific monoclonal antibody heteropolymer (HP). Lack of a functional homologue to CR1 on mouse erythrocytes has made it difficult to study HP-dependent clearance of pathogens in small animals. We have developed a transgenic mouse that expresses human CR1 on erythrocytes. CR1 antigen is of appropriate size and in a clustered distribution as confirmed by immunoblotting and fluorescence microscopy, respectively. HP that immobilized bacteriophage PhiX174 prototype pathogen to erythrocyte CR1 of the transgenic mice increased the rate of clearance of the virus compared with HP that bound bacteriophage, but not CR1. This transgenic mouse model will allow evaluation of different HPs for their in vivo efficacy and potential as human therapeutics.

Rituximab inhibits the in vivo primary and secondary antibody response to a neoantigen, bacteriophage phiX174.

The response to primary immunization in patients treated with Rituximab (RIT) is not clear. We studied the in vivo antibody response of chronic renal failure (CRF) patients to the neoantigen bacteriophage phiX174 given alone or after ablation with RIT. Eighteen CRF subjects received two immunizations with phiX174 separated by 6 weeks. Nine subjects received a single dose of RIT. The intensity and immunoglobulin isotype of the antibody response (K(v)) were measured post-infusion. In addition, three subjects previously immunized and treated with RIT underwent a third and fourth immunization with phiX174 and a tetanus control 2 years later. RIT significantly decreased peak K(v) responses when compared to both historic non-CRF controls and to CRF subjects. CRF itself decreased peak K(v) responses compared to non-CRF controls. Percent-ratio of anti-phage IgM to IgG was significantly decreased in RIT treated subjects. One of three subjects treated with RIT was found to have developed a partial B cell tolerance to phiX174 administration 2 years later. RIT decreases antibody production and isotype switching to neoantigens and might be useful to prevent antibody response to therapeutic drugs and to newly transplanted organs.

CD4+ T-cell-directed antibody responses are maintained in patients with psoriasis receiving alefacept: results of a randomized study.

BACKGROUND: Alefacept, human LFA-3/IgG(1) fusion protein, selectively reduces memory-effector (CD45RO(+)) T cells, a source of the pathogenic mediators of psoriasis. OBJECTIVE: To evaluate the effect of alefacept on immune function, T-cell-dependent humoral responses to a neoantigen (PhiX174) and recall antigen (tetanus toxoid) were assessed. METHODS: Patients with psoriasis were randomized to the control group or to receive alefacept (7.5 mg intravenously weekly for 12 weeks). The alefacept group received PhiX174 immunizations at weeks 6, 12, 20, and 26 and tetanus toxoid at week 21; control subjects received PhiX174 at weeks 6 and 12 and tetanus at week 10. RESULTS: Mean anti-PhiX174 titers were comparable in both groups. There was no difference in the percentage of responders (anti-PhiX174 IgG >/=30% of the total anti-PhiX174) between the alefacept group and the control group (86% and 82%, respectively; P =.73). The percentage of patients with anti-tetanus toxoid titer increases >/=2 times baseline also was similar (alefacept, 89%; control 91%). CONCLUSION: A single 12-week course of alefacept did not impair primary or secondary antibody responses to a neoantigen or memory responses to a recall antigen. The selective immunomodulatory effect of alefacept against a potentially pathogenic T-cell subset is associated with maintenance of a significant aspect of immune function (antibody response) to fight infection and respond to vaccinations.

The antibody response to bacteriophage is linked to the lymphopenia gene in congenic BioBreeding rats.

Congenic BioBreeding (BB) rats, homozygous for the autosomal lymphopenia (Lyp) gene (Lyp/Lyp), heterozygous (Lyp/+), or wild-type (+/+), were immunized with the T cell-dependent bacteriophage PhiX174 to determine effects of Lyp on primary and secondary antibody responses. The primary PhiX174 antibody response did not differ between the three different genotypes. In contrast, the secondary immune response, expressed as the peak neutralizing titer, was markedly reduced in Lyp/Lyp (9.9+/-3.2; mean value+/-S.E.M. for seven rats) compared to both Lyp/+ (51+/-12; n=13; P=0.006) and +/+ (100+/-20; n=7; P=0.004) BB rats. We suggest that the secondary antibody response to the T cell-dependent neoantigen PhiX174 is linked in a recessive manner to genetic factor(s) in the Lyp gene region.

Evaluation of CD4+ T cell function In vivo in HIV-infected patients as measured by bacteriophage phiX174 immunization.

Bacteriophage phiX174 immunization was used to measure CD4(+) T cell function in vivo in human immunodeficiency virus (HIV)-infected patients across all disease stages. Function was evaluated by measuring the ability of T cells to provide help to B cells in antibody production, amplification, and isotype switching. A total of 33 patients and 10 controls received 3 bacteriophage phiX174 immunizations 6 weeks apart. The patients' responses regarding bacteriophage-specific total antibody titers and IgG titers were quantitatively and qualitatively inferior to the controls' responses. Overall, 7 of 33 patients had normal T cell function. Baseline CD4 counts provided the strongest correlation with total antibody and IgG titers. HIV RNA had a weaker association with responses but had some predictive power among patients with a CD4 count >200 cells/microL. Bacteriophage phiX174 immunization seems to be a useful tool for measuring immune function in vivo, which suggests that most HIV-infected patients may have abnormal CD4(+) T cell function despite adequate antiretroviral treatment.

Progressive specific immune attrition after primary, secondary and tertiary immunizations with bacteriophage phi X174 in asymptomatic HIV-1 infected patients.

BACKGROUND: Antibody responses to immunization are often compromised in patients infected by HIV-1, and the use of childhood immunization in affected children is controversial. We investigated whether multiple immunizations with a T cell-dependent neoantigen, bacteriophage phiX174, induce selective immune attrition and post-vaccination viremia. METHODS: Seventeen asymptomatic, antiretroviral therapy-naïve HIV-1-infected patients with a CD4 cell count of 450 cells/microl or greater were immunized in 1990/1991 with three intravenous doses of bacteriophage phiX174. Group 1 received zidovudine (ZDV) during the primary and secondary immunization. Group 2 received ZDV exclusively during the tertiary immunization. Bacteriophage-specific antibodies of the IgM and IgG class, lymphocyte phenotypes (CD4+, CD8+, CD4+DR+, CD8+DR+, CD4+CD45RO+ and CD4+45RA+, CD4+CD45RO+DR+) and HIV-1 plasma viremia were measured sequentially. RESULTS: In both patient groups the primary, secondary and tertiary antibody responses, as expressed by geometric mean antibody titres and IgM to IgG switch, were impaired. Booster immunizations resulted in a progressive attrition of specific antibody responses to bacteriophage. Antibodies to tetanus toxoid remained stable. The HIV-1 viral loads, which were evaluated in archived specimens from eight patients, increased after immunization but returned to baseline approximately 4 weeks later. The humoral immune attrition and increases in plasma viremia were blunted by concomitant short courses of ZDV. DISCUSSION: Multiple boosters of immunizations in asymptomatic treatment-naive HIV-1-infected patients may result in a specific immune attrition and vaccine-induced viremia. Short-term monotherapy with ZDV may have blunted these adverse effects. Hyperimmunization of HIV-1-infected patients may be detrimental unless accompanied by antiretroviral therapy.

Transfer of immune complexes from erythrocyte CR1 to mouse macrophages.

We are developing a potential therapeutic approach for removing pathogens from the circulation of primates in which the pathogen is bound to the complement receptor (CR1) on E using a bispecific mAb complex, a heteropolymer (HP). We have used mAb this approach to demonstrate that cleared prototype pathogens are localized to, phagocytosed in, and destroyed in the liver. Extension of this work to a clinical setting will require a detailed understanding of the mechanism by which the E-bound immune complex substrates are transferred to fixed tissue macrophages in the liver, the transfer reaction. Therefore, we examined an in vitro system to study this process using bacteriophage phiX174 as a model pathogen. E containing phiX174 (bound via an anti-CR1/anti-phiX174 HP) were incubated with P388D1 murine macrophages, and the two cell types were separated by centrifugation through Ficoll. Both E and macrophages were then probed and analyzed by RIA or flow cytometry. The results indicate that all three components of the E-bound IC (phiX174, HP, and CR1) were removed from the E and internalized by the macrophages. We found that transfer requires the Fc portion of IgG, because little transfer of phiX174 occurs when it is bound to E CR1 using a HP containing only Fab fragments. These findings, taken in the context of other studies, suggest a general mechanism for the transfer reaction in which Fc receptors facilitate close juxtaposition of the macrophage to the E-bound IC which then allows a macrophage-associated protease to cleave CR1. The released IC are then internalized and processed by the macrophages.

Infusion of bispecific monoclonal antibody complexes into monkeys provides immunologic protection against later challenge with a model pathogen.

Heteropolymers (HP), bispecific mAbs which bind target pathogens to primate erythrocytes via complement receptor 1, facilitate clearance of pathogens from the bloodstream by targeting them for destruction in the liver without causing lysis or clearance of the erythrocytes. We show that when HP prepared with mouse IgG are intravenously infused into monkeys one or more times prior to exposure to a prototype pathogen, they bind to erythrocytes and remain in the circulation long enough to act as "sentinels," preventing pathogen invasion of the bloodstream. The effectiveness of HP as sentinels is limited both by the monkey's immune response to the HP and, prior to the immune response, by a gradual loss of the HP from monkey erythrocytes over a period of 1 week, and we have investigated possible causes of this HP loss. In overview, our results suggest that HP prepared with mouse IgG are able to effectively function as sentinels for a minimum of 4 days and, after repeat infusion, possibly for up to 2 weeks.

Mutations of the CD40 ligand gene and its effect on CD40 ligand expression in patients with X-linked hyper IgM syndrome.

X-linked hyper IgM syndrome (XHIM) is a primary immunodeficiency disorder caused by mutations of the gene encoding CD40 ligand (CD40L). We correlated mutations of the CD40L gene, CD40L expression, and the clinical manifestations observed in XHIM patients from 30 families. The 28 unique mutations identified included 9 missense, 5 nonsense, 9 splice site mutations, and 5 deletions/insertions. In 4 of 9 splice site mutations, normally spliced and mutated mRNA transcripts were simultaneously expressed. RNase protection assay demonstrated that 5 of 17 mutations tested resulted in decreased levels of transcript. The effect of the mutations on CD40L expression by activated peripheral blood mononuclear cells (PBMC) and T-cell lines or clones was assessed using one polyclonal and four monoclonal antibodies and a CD40-Ig fusion protein. In most patients, the binding of at least one antibody but not of CD40-Ig was observed, suggesting nonfunctional CD40L. However, activated PBMC from three patients and activated T-cell lines from two additional patients, each with different genotype, bound CD40-Ig at low intensity, suggesting functional CD40L. Thus, failure of activated PBMC to bind CD40-Ig is not an absolute diagnostic hallmark of XHIM and molecular analysis of the CD40L gene may be required for the correct diagnosis. Patients with genotypes resulting in diminished expression of wild-type CD40L or mutant CD40L that can still bind CD40-Ig appear to have milder clinical consequences.

Diminished expression of CD40 ligand may contribute to the defective humoral immunity in patients with MHC class II deficiency.

Major histocompatibility complex (MHC) class II deficiency (bare lymphocyte syndrome, BLS) is a rare primary immunodeficiency classified as a subgroup of severe combined immunodeficiency. We studied T and B lymphocyte function by examining the CD40 ligand/CD40 system in three BLS patients from two unrelated families. CD40 ligand expression by maximally activated BLS T cells was diminished. This abnormality may represent immunological naïveté rather than a general T cell defect, since expression of activation marker CD69 and proliferative responses to PHA or anti-CD3 were normal, and BLS T cells primed and restimulated in vitro expressed normal amounts of CD40 ligand. BLS B cells proliferated and produced IgE if stimulated with anti-CD40 or soluble CD40 ligand and IL-4. Activation of BLS B cells with soluble CD40 ligand and IL-4 induced normal expression of activation markers, although MHC class II expression remained absent. Depressed antibody titers, lack of amplification and failure to undergo isotype switching in response to immunization with bacteriophage phi x 174 demonstrated defective T cell help. We conclude that BLS B cells are functionally normal if appropriately stimulated, and that the defective humoral immunity observed may be related to diminished expression of CD40 ligand on BLS T cells.

Bispecific monoclonal antibody complexes facilitate erythrocyte binding and liver clearance of a prototype particulate pathogen in a monkey model.

We used Anger camera imaging in a monkey model to investigate the organ localization of a prototype particulate pathogen, 131I-labeled bacteriophage phi X174, after it was bound to the primate erythrocyte complement receptor and then cleared from the circulation. This 131I-labeled phi X174 was infused into the circulation of an immunized monkey, and the nascently formed immune complexes showed rapid and quantitative binding to erythrocytes via the immune adherence reaction (complement-mediated binding). Alternatively, phi X174 was infused into the circulation of a naive animal, and then cross-linked bispecific mAb complexes (heteropolymers, anti-CR1 x anti-phi X174) were infused into the circulation. The infused heteropolymers also facilitated rapid and quantitative binding of phi X174 to erythrocytes. In both cases, after a short lag period, the erythrocyte-bound phi X174 was rapidly cleared from the circulation, and the vast majority of the radiolabel was cleared to the liver, with a small amount clearing to the spleen. Further liver imaging confirmed that within 24 h most of the bacteriophage previously cleared to the liver via the heteropolymer system was phagocytosed and destroyed. The findings in this model system provide additional evidence for the potential utility of heteropolymers to facilitate the safe and rapid clearance of blood-borne pathogens as a potential treatment for infectious diseases.