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Patients on immunosuppressive drugs have been excluded from studies of SARS-CoV-2 mRNA vaccines’ (mRNA-BNT162b2 and mRNA-1273) clinical trials that resulted in their Emergency Use Authorization in the USA and Europe. Since the initiation of the global vaccination campaign, it became apparent that patients on immunosuppressive drugs may not generate optimal immune response following vaccination.1 Prednisone and mycophenalate are potent inhibitors of immune responses. Prednisone acts at many levels of immunity including the innate and adaptive responses, whereas mycophenalate mainly targets T and B cells.2 These and other immunosuppressive drugs used to treat patients with autoimmune disorders and organ transplant patients were shown to significantly curtail antibody responses following SARS-CoV-2 mRNA vaccines.3–5 We report on a 75-year-old male patient with an underlying autoimmune disorder, myasthenia gravis, which was controlled after receiving high doses of prednisone and mycophenalate for 9 months. At his first SARS-CoV-2 vaccination, he was receiving 7.5 mg prednisone on alternate days and 3 g mycophenalate daily (figure 1A). He received two doses of Moderna mRNA-1273 vaccine with a 4-week interval between doses. Neutralisation titres were measured using a pseudovirus neutralisation assay (PsVNA) as described previously.6 No SARS-CoV-2 neutralising antibodies were detected in plasma at 4 weeks after the second mRNA-1273 …

The most challenging complication associated with Factor VIII (FVIII) replacement therapy is the development of neutralizing anti-drug antibodies, or inhibitors, which occur in 23-35% of severe (FVIII level via neonatal Fc receptor (FcRn) binding, other Fc-mediated interactions, including engagement with Fc gamma receptors (FcγR), may have immunological consequences. Several case reports and retrospective analyses suggest that rFVIIIFc offers superior outcomes with respect to ITI compared to other FVIII products. Previously we and others demonstrated rFVIIIFc interactions with activating FcγRIIIA/CD16. Here, we investigated if rFVIIIFc activates natural killer (NK) cells via CD16. We demonstrated rFVIIIFc signaling via CD16 independent of Von Willebrand Factor (VWF):FVIII complex formation. We established that rFVIIIFc potently activated NK cells in a CD16-dependent fashion resulting in IFNγ secretion and cytolytic perforin and granzyme B release. We also demonstrated an association between rFVIIIFc-mediated NK cell IFNγ secretion levels and the high-affinity (158V) CD16 genotype. Furthermore, we show that rFVIIIFc-activated CD16+ NK cells were able to lyse a B-cell clone (BO2C11) bearing an anti-FVIII B-cell receptor in an antibody-dependent cellular cytotoxicity (ADCC) assay. These in vitro findings provide an underlying molecular mechanism that may help explain clinical case reports and retrospective studies suggesting rFVIIIFc may be more effective in tolerizing HA patients with anti-FVIII inhibitors compared to FVIII not linked to Fc. Our in vitro findings suggest a potential use of Fc-fusion proteins acting via NK cells to target antigen-specific B-cells, in the management of unwanted immune responses directed against immunogenic self-antigens or therapeutic protein products.

The most challenging complication associated with Factor VIII (FVIII) replacement therapy is the development of neutralizing anti-drug antibodies, or inhibitors, which occur in 23-35% of severe (FVIII level

Background Immunoglobulins are widely used across multiple therapeutic areas such as immunodeficiency syndromes, infection and autoimmune diseases. The pharmacokinetics (PK) of immunoglobulins are well characterized in adults, but very little is known about the PK of immunoglobulins in neonates and infants. Objective The objective of the present study was to characterize the PK of Gammagard, an immunoglobulin, in very low birth weight preterm neonates. Method Gammagard concentration-time data from very low birth weight neonates (bodyweight range 0.78–1.38 kg, n = 20) following intravenous administration of 500 mg/kg and 750 mg/kg were obtained from the literature. The data were analyzed with and without baseline correction using extensive blood samples (8 blood samples). Model-independent (non-compartmental) analysis was used to characterize the PK of Gammagard. Results Based on uncorrected baseline concentration-time data, the clearance and half-life of Gammagard were 3.1 ± 0.7 mL/day and 22 ± 6 days, respectively. Based on corrected baseline concentration-time data, the clearance and half-life of Gammagard were 20.2 ± 7.4 mL/day and 5.3 ± 2.2 days, respectively. Conclusion The dose of immunoglobulins should be adjusted based on the PK of baseline corrected rather than baseline uncorrected profiles because baseline corrected PK parameters especially half-life reconciles with PK principles.

FactorVIII Fc-fusion (rFVIIIFc) is an enhanced half-life antihemophilic factor usedin replacement therapy of hemophilia A. Fc-fusion is a successful protein bio-engineering platform technology. In addition to enhancement of plasmahalf-life via neonatal Fc receptor (FcRn) binding, other Fc-mediatedinteractions, including engagement with Fc gamma receptors (FcγR), may haveimmunological consequences. Previouslywe and others demonstrated rFVIIIFc interactions with activatingFcγRIIIA/CD16. Here, we investigated ifrFVIIIFc activates natural killer (NK) cells via CD16. We demonstrated rFVIIIFc signaling via CD16independent of Von Willebrand Factor (VWF):FVIII complex formation. We established that rFVIIIFc potentlyactivated NK cells in a CD16-dependent fashion resulting in IFNγ secretion andcytolytic perforin and granzyme B release. We also demonstrated an association between rFVIIIFc-mediated NK cellIFNγ secretion levels and the high affinity (158V) CD16 genotype. Furthermore, we show that rFVIIIFc-activatedCD16+ NK cells were able to lyse a B-cell clone bearing ananti-FVIII BCR (BO2C11) in an antibody-dependent cellular cytotoxicity (ADCC)assay. These findings provide anunderlying molecular mechanism that may help explain clinical case reports andretrospective studies that suggest rFVIIIFc may be more effective in tolerizinghemophilia A patients with anti-FVIII inhibitory antibodies compared to FVIIInot linked to Fc. Our findings suggest apotential use of Fc-fusion proteins acting via NK cells to target B-cells, inthe management of unwanted immune responses directed against immunogenicself-antigens or therapeutic protein products.

Immunoglobulins (IGs) are widely used for the treatment of immunodeficiency syndromes and several autoimmune diseases. In neonates, IGs have been used for the treatment of alloimmune thrombocytopenia, in neonatal infections and in the rare cases of neonatal Kawasaki disease. This review aims to examine the various dosing regimens of IGs following intravenous (IV) and subcutaneous (SC) administration, pharmacokinetics (PK) of IGs, and the importance of trough values for the prevention of infections in patients with primary immune deficiency (PID). The review also focuses on the mechanism of catabolism of IGs and the impact on the half-life of IGs. Data and reviews were obtained from the literature and the FDA package inserts. The authors suggest that for dosing, the PK of IGs should be evaluated on the baseline-corrected concentrations since this approach provides an accurate estimate of half-life and clearance of IGs. We also suggest employing clearance as a primary PK parameter for dosing determination of IGs. We suggest that IV dosing would be more effective if given more frequently to adjust for the increased clearance at high doses and because the baseline-corrected half-life is much shorter than the baseline-uncorrected half-life. Regarding SC administration, the dose should be adjusted based on the absolute bioavailability (determined against IV dosing) of the product. Finally, we highlight clinical and PK data gaps for optimum and individualized dosing of IGs.

To study bacterial co-infection following 1918 H1N1 influenza virus infection, mice were inoculated with the 1918 influenza virus, followed by Streptococcus pneumoniae (SP) 72 h later. Co-infected mice exhibited markedly more severe disease, shortened survival time and more severe lung pathology, including widespread thrombi. Transcriptional profiling revealed activation of coagulation only in co-infected mice, consistent with the extensive thrombogenesis observed. Immunohistochemistry showed extensive expression of tissue factor (F3) and prominent deposition of neutrophil elastase on endothelial and epithelial cells in co-infected mice. Lung sections of SP-positive 1918 autopsy cases showed extensive thrombi and prominent staining for F3 in alveolar macrophages, monocytes, neutrophils, endothelial and epithelial cells, in contrast to co-infection-positive 2009 pandemic H1N1 autopsy cases. This study reveals that a distinctive feature of 1918 influenza virus and SP co-infection in mice and humans is extensive expression of tissue factor and activation of the extrinsic coagulation pathway leading to widespread pulmonary thrombosis. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Background Thrombotic events (TEs) are rare and serious adverse events after administration of immune globulin (IG) products. Our study evaluated the occurrence of same-day TEs for different IG products and ascertained potential risk factors. Study Design and Methods This retrospective cohort study utilized HealthCore's Integrated Research Database (HIRD) to assess individuals exposed to IGs during 2008 to 2012. IG products were identified using recorded procedure codes and TEs were ascertained using ICD-9-CM diagnosis codes. The unadjusted same-day TE rates (per 1000 persons exposed) were estimated overall and by IG products, age, and sex. Multivariable logistic regression analyses were used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for same-day TEs by IG products. Results Of 14,944 individuals exposed to IG products, 233 (15.6 per 1000 persons) had TE diagnosis code(s) recorded on the same-day as the IG exposure. Compared to Gammagard Liquid, Gammaplex (OR, 20.96; 95% CI, 2.45-179.33) and Vivaglobin (OR, 2.74; 95% CI, 1.19-6.32) users had a significantly increased same-day TE risk. Elevated, but nonsignificant TE risks were identified for Octagam, Gamunex, Privigen, and Lyophilized IG(s). An increased TE risk was also found with older age (≥45 years), prior TEs, and other health conditions. Conclusion Our claims-based cohort study suggests a potentially elevated TE risk with different IG products and shows importance of recipient factors such as older age, previous TE, hypercoagulable state(s), and other health conditions. The results of this study suggest the need for continuous evaluation of procoagulant activity and manufacturing processes for IG products to further assure their safety.

Essentials Fc-fusion increases a therapeutic's half-life, but FcγR interactions may impact immunogenicity. Species-specific Fc-FcγR interactions allow for mechanistic in vivo studies using mouse models. Fc fusion modulates the immune response to factor IX in hemophilia B mice by eliciting Th1 bias. This model could inform future studies of IgE-associated anaphylaxis in hemophilia B patients. SummaryBackground 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) that 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 Although 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 with the hFIX-treated group. Additionally, differences in plasma cytokine levels and in vitro CD4+ T-cell responses suggest that whereas 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.

The infusion of plasma-derived or recombinant factors to treat bleeding disorders such as hemophila A and B is a success story in the management of a chronic disease. The effectiveness of this approach is however limited by challenges with adverse effects of treatment. The most notable of these are the development of inhibitory antibodies that target the protein therapeutic. The current standard of care for management of hemophiliacs is prophylactic treatment that includes frequent infusions of a Factor VIII product. Failure to comply with the prophylactic regimen is a major hurdle in the management of these patients. We discuss here more recent findings that argue for a pharmacogenetic approach to understanding (and eventually circumventing) immunogenicity. We also review strategies used to bioengineer coagulation factors to extend the half-lives of coagulation proteins. The rapid progress in the last few years to bioengineer coagulation factors in different ways to attain this goal is described. Finally, novel technologies and potential products are emerging that utilize synthetic molecules in lieu of replacement proteins obviating the limitations associated with replacement therapies.

BACKGROUND: Thrombotic events (TEs) are rare but often serious adverse events that could occur after administration of immune globulin (IG) products. Our study objective was to assess occurrence of recorded TEs after administration of different US-licensed IG products and investigate potential risk factors using a large administrative database. STUDY DESIGN AND METHODS: This is a retrospective claims-based cohort study of individuals exposed to IG products from January 1, 2008, through September 30, 2010, using HealthCore's Integrated Research Database, a longitudinal health care database. IG products were identified by recorded Healthcare Common Procedure Coding System codes. TEs were ascertained via International Classification of Diseases, Ninth Revision, Clinical Modification diagnosis codes. Logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for same-day TEs by IG product, while controlling for confounders. RESULTS: Of 11,785 individuals exposed to IG products in the study period, 122 (1%) had TE(s) recorded on the same day as IG administration. TE rates per 1000 persons exposed ranged from 6.1 to 20.5 for different IG product groups. Vivaglobin users had an increased same-day TE risk compared to reference Gammagard Liquid users (OR, 3.56; 95% CI, 1.54-8.23). An increased TE risk was also found with older age (≥45 years), prior TE(s), and hypercoagulable state(s). CONCLUSION: The study suggests potentially elevated TE rates for different IG products, including subcutaneous. It also identifies important recipient TE risk factors and suggests that risk–benefit profiles should be weighed before IG administration. The observed differences may be due to various factors such as dosage, administration rates, and product manufacturing processes that warrant further evaluation.

Human alpha-1-proteinase inhibitor is a well-characterized protease inhibitor with a wide spectrum of anti-protease activity. Its major physiological role is inhibition of neutrophil elastase in the lungs, and its deficiency is associated with progressive ultimately fatal emphysema. Currently in the US, only plasma-derived human alpha-1-proteinase inhibitor is available for augmentation therapy, which appears to be insufficient to meet the anticipated clinical demand. Moreover, despite effective viral clearance steps in the manufacturing process, the potential risk of contamination with new and unknown pathogens still exists. In response, multiple efforts to develop recombinant versions of human alpha-1-proteinase inhibitor, as an alternative to the plasma-derived protein, have been reported. Over the last two decades, various systems have been used to express the human gene for alpha-1-proteinase inhibitor. This paper reviews the recombinant versions of human alpha-1-proteinase inhibitor produced in various hosts, considers current major safety and efficacy issues regarding recombinant glycoproteins as potential therapeutics, and the factors that are impeding progress in this area(1).

The role of activated CD8+ T cells in shaping the dynamics of in vivo antigen presentation and immune responses is a subject receiving more attention. We studied whether cytotoxic T lymphocyte (CTL) would limit antibody responses by targeting antigen-specific B cells. A modified in vivo CTL assay was developed and used herein to demonstrate cytotoxicity in vivo, and to show that antigen-specific B cells that process exogenous antigen and present peptide in association with MHC class I can be the targets of CD8+ T cells. B cells from C57BL/6 mice immunized with ovalbumin (OVA)/alum were pulsed with OVA in vitro, and transferred into C57BL/6 recipient mice that had been immunized with vaccinia virus expressing SIINFEKL minigene to generate CD8+ CTL against K(b)/SIINFEKL. OVA-pulsed B220+ B cells from OVA-immunized mice were killed to a greater extent than B220+ B cells from naïve mice (28+/-20% versus 12+/-16%, p=0.0042). However, mice receiving vaccinia-SIINFEKL and generating CTL, did not appear to target endogenous B cells, since both primary and secondary antibody responses to OVA were unaffected. Our findings indicate that CTL responses to the protein antigen do not interfere with endogenous B cell responses, even though exogenous B cells expressing the CTL epitope can be efficiently lysed.

Optimal generation of cytotoxic T cell (CTL) responses continues to be a challenge in the production of vaccines against pathogens such as HIV-1, in part because it is difficult to introduce soluble protein antigens (Ag) into the MHC class I pathway. Using heat-killed Brucella abortus (HKBA) as an adjuvant and ovalbumin (ova) protein as an Ag, we demonstrated that a high dose of Ag was required for systemic and effective CTL. In an adoptive transfer model, primary and secondary ova-specific OT-1 CD8 cell expansion by HKBA plus high dose of ova were partially CD4 T cell-dependent. Interestingly, primary stimulation with HKBA plus ova allowed effective secondary stimulation with ova alone that was equivalent to HKBA plus ova in terms of IFN-γ production from Ag-specific CD8 cells. Thus a combination of adequate Ag dose, and selection of appropriate adjuvants can meet the threshold not only for primary effective CTL responses to soluble protein Ags but for secondary CTL responses following stimulation with protein Ag alone.

The most common infections in primary immune deficiency disease (PIDD) patients involve encapsulated bacteria, mainlyHaemophilus influenzaetype b (Hib) andStreptococcus pneumoniae(pneumococcus). Thus, it is important to know the titers of Hib- and pneumococcus-specific antibodies that are present in immune globulin (Ig) intravenous (IGIV) preparations used to treat PIDD. In this study, seven IGIV preparations were tested by enzyme-linked immunosorbent assay and opsonophagocytic activity for antibody titers to the capsular polysaccharides of Hib and five pneumococcal serotypes. Differences in Hib- and pneumococcus-specific antibody titer were observed among various IGIV preparations, with some products having higher- or lower-than-average titers. Opsonic activity also varied among preparations. As expected, IgG2 was the most active subclass of both binding and opsonic activity except against pneumococcal serotype 6B where IgG3 was the most active. This study determines antibody titers against capsular polysaccharides of Hib and pneumococcus in seven IGIV products that have been shown to be effective in reducing infections in PIDD patients. As donor antibody levels and manufacturing methods continue to change, it may prove useful from a regulatory point of view to reassess IGIV products periodically, to ensure that products maintain antibody levels that are important for the health of IGIV recipients.

HIV vaccine testing in primates is an important method for determining the possibility of vaccine benefit in humans. Goals of HIV-1 vaccination include establishing neutralizing antibodies and a strong CD8(+) T-cell response. We tested a novel vaccine conjugate for its ability to elicit relevant immune responses to HIV proteins and peptides in rhesus macaques. A neutralizing epitope, V3 loop peptide from HIV-1 envelope, was coupled to heat-inactivated Brucella abortus (V3-HKBA). Rhesus macaques were immunized with this conjugate in the anterior thigh. After two immunizations V3-specific antibodies were found in the sera and at mucosal sites. Neutralizing activity of these antibodies was demonstrated by syncytia inhibition assays. Cellular immune recall responses were demonstrated by antigen-specific induction of interferon-gamma and Regulation on Activation Noraml T Cell Expressed and Secreted (RANTES) secretion in vitro. These results confirm and extend preliminary studies in mice that suggest HKBA is an effective carrier that promotes neutralizing antibody secretion at relevant mucosal sites, as well as cellular immune responses that are correlated with viral protection.

Fusion of the IgG1 constant region domain (Fc) to therapeutic proteins increases the plasma half-life of drugs via binding to the neonatal Fc receptor (FcRn). However, potential interactions with Fc gamma-receptors (FcγR) expressed by immune cells may effect the immunogenicity of these drugs. The coagulation factors VIII (FVIII) and IX (FIX) for the treatment of hemophilia A & B (HA & HB) are the only drugs available with and without Fc fusion and offer an opportunity to experimentally study the effect of Fc fusion. Anti FVIII & FIX antibodies (ADAs) and rare cases of anaphylaxis in HB patients, are serious safety concerns. Using a mouse model for HB, we compared the immune responses to infusions of recombinant human FIX (hFIX) and hFIX fused to mouse IgG2a-Fc (hFIX-mFc). The mFc allowed species-specific Fc-FcγR interactions and prevented immune response to the foreign hFc. Treatment with hFIX-mFc caused earlier onset of anti-FIX IgG and elicited higher FIX-neutralizing antibody levels. Treatment with hFIX triggered Th2 biased immunity i.e., elevated plasma IgE titers, and Th2 cytokine profile of FIX-specific CD4 T cells and a few anaphylactic events. The fusion of Fc altered the response to a Th1 type with increased plasma IFN-γ levels, and IFN-γ secretion by effector CD4 T cells. hFIX-mFc binds to soluble FcγRs and FcγRs on natural killer cells (which also secrete IFNγ) and antigen presenting cells (dendritic cells, and macrophages source of IL-12). We show that Fc fusion enhances the presentation of hFIX to CD4 T cells. We propose that high plasma availability and targeting of APC provide plausible mechanisms by which the Fc fusion may modulate the CD4 T cell response to hFIX towards a Th1 like response, thus avoiding allergic responses.

Fusing the IgG1 constant region (Fc-domain) to therapeutic proteins increases their plasma half-life via neonatal Fc receptor (FcRn) binding. However, potential interactions with other Fc-binding molecules including complement C1q and Fc gamma receptors (FcγR) can have immunological consequences. Our previous studies in a mouse model suggest that engagement of the Fc-Factor IX (FIX) to FcγR on APC and NKT cells could alter the immunogenicity of the FIX moiety. Here, we conducted a comparative study of FDA-approved Fc-fusion proteins to assess their potential for C1q binding as well as FcγR binding and signaling. Binding of soluble drug to C1q was measured by ELISA, and FcγR binding and signaling was evaluated using BW5147:FcγR-ζ reporter cell lines. We demonstrate that rFIXFc and rFVIIIFc bound C1q as well as activating and inhibitory FcγRs (I, IIA, IIB, IIIA). These therapeutic proteins also signaled via FcγRIIIA, and to a lesser extent via FcγRI and FcγRIIB. Other Fc-fusion drugs (TNFR-Fc; CTLA4-Fc) bound FcγRI (TNFR-Fc also bound FcγRIIIA), but did not result in FcγR signaling. As predicted, a control anti-CD20 monoclonal antibody modified for enhanced FcγRIIIA engagement, bound and signaled via FcγRIIIA, while the parent molecule did not; supporting our use of BW5147: FcγR-ζ reporter cells for the assessment of Fc:FcγR interactions. Our studies show that Fc-fusion drugs have distinct C1q/FcγR binding and signaling properties. Moreover, FcγR binding does not predict signaling. Our studies suggest that Fc-FcγR engagement can influence the immunogenicity to Fc-fusion drugs as both fusion partners influence this interaction. Future investigations will elucidate the underlying molecular mechanisms.

The platform technology of fragment crystallizable (Fc) fusion, in which the Fc region of an antibody is genetically linked to an active protein drug, is among the most successful of a new generation of bioengineering strategies. Immunogenicity is a critical safety concern in the development of any protein therapeutic. While the therapeutic goal of generating Fc-fusion proteins has been to extend half-life, there is a critical mass of literature from immunology indicating that appropriate design of the Fc component has the potential to engage the immune system for product-specific outcomes. In the context of Fc-fusion therapeutics, a review of progress in understanding Fc biology suggests the prospect of engineering products that have an extended half-life and are able to modulate the immune system.

Naive T helper cells can differentiate into one of two subsets, known as Th1 or Th2, depending on the context of the initial stimulation they receive (10). In the presence of interleukin-12 (IL-12) Th1 cells, which secrete IL-2, gamma interferon (IFN-γ), and tumor necrosis factor beta (TNF-β), develop. On the other hand, induction of IL-4 favors the development of Th2 cells, which produce IL-4, IL-5, IL-6, IL-10, and IL-13 (11, 25). Antibody class switching in the mouse is influenced by T-cell cytokines such that IFN-γ promotes secretion of immunoglobulin G2a (IgG2a) antibodies, and IL-4 promotes secretion of IgG1 and IgE (1, 4). T-cell subsets are also capable of cross-regulation: IFN-γ inhibits the actions of IL-4 on resting B cells, including secretion of IgE and IgG1 isotypes (18). IL-4, on the other hand, down-regulates the secretion of IFN-γ by CD4+ T cells (14). Antigen-specific responses have not been studied in detail at early stages of immune development. Host protection against intracellular infections, such as Brucella abortus infections, involves a Th1-like response (9). Heat-killed B. abortus (HKBA) has been shown to up-regulate the secretion of IL-12 and IFN-γ (2, 12, 21) and is considered a strong Th1 stimulus (2, 19). We have recently demonstrated that a single injection of B. abortus, when injected together with ovalbumin adsorbed to alum (OVA-A) into adult mice, has the ability to abolish antigen-specific IgE-mediated allergic responses to repeated OVA-A challenge and to increase production of IgG2a in vivo (12, 13). This effect of B. abortus on isotype switching correlated with an increase in IFN-γ and a decrease in IL-4-secreting-cells (12). Thus, B. abortus has the ability to alter the antigen-specific cytokine profile from Th2- to Th1-like when injected together with OVA-A in adult mice. This has implications for mounting effective immune responses against B. abortus, as well as enabling B. abortus to be used as a vaccine adjuvant or carrier in situations where Th1 reactivity is beneficial. In this report we examine the ontogeny of this Th1-to-Th2 switch in young mice using OVA conjugated to HKBA (HKBA-OVA) as a Th1 stimulus. The results have implications for immunization and protection against bacterial infections. In addition, they provide insights into approaches that could prevent allergic disease.

Heat-killedBrucella abortus(HBa) has been proposed as a carrier for therapeutic vaccines for individuals with immunodeficiency, due to its abilities to induce interleukin-2 (IL-2) and gamma interferon (IFN-γ) in both CD4+and CD8+T cells and to upregulate antigen-presenting cell functions (including IL-12 production). In the current study, we investigated the ability of HBa or lipopolysaccharide isolated from HBa (LPS-Ba) to elicit β-chemokines, known to bind to the human immunodeficiency virus type 1 (HIV-1) coreceptor CCR5 and to block viral cell entry. It was found that human peripheral blood mononuclear cells secreted β-chemokines following stimulation with HBa, and this effect could not be blocked by anti-IFN-γ neutralizing antibodies. Among purified T cells, macrophage inflammatory protein 1α and 1β (MIP-1α and MIP-1β, respectively) secretion was observed primarily in human CD8+T cells. The kinetics of β-chemokine induction in T cells were slow (3 to 4 days). The majority of β-chemokine-producing CD8+T cells also produced IFN-γ following HBa stimulation, as determined by triple-color intracellular staining. A significant number of CD8+T cells contained stored MIP-1β that was released after HBa stimulation. Both HBa and LPS-Ba stimulated high levels of MIP-1α and MIP-1β production in elutriated monocytes and even higher levels in macrophages. In these cells, β-chemokine mRNA was upregulated within 30 min and proteins were secreted within 4 h of stimulation. The monocyte- and macrophage-derived β-chemokines were sufficient to block CCR5-dependent HIV-1 envelope-mediated cell fusion. These data suggest that, in addition to the ability of HBa to elicit antigen-specific humoral and cellular immune responses, HBa-conjugated HIV-1 proteins or peptides would also generate innate chemokines with antiviral activity that could limit local viral spread during vaccination in vivo.

Down-regulation of the Th2-like response induced by ovalbumin-alum (OVA/alum) immunization by heat-killed Brucella abortus was not reversed by anti-IL-12 antibody treatment or in gamma interferon (IFN-γ) knockout mice, suggesting that induction of Th1 cytokines was not the only mechanism involved in the B. abortus -mediated inhibition of the Th2 response to OVA/alum. The focus of this study was to determine whether an alternative pathway involves alteration in expression of costimulatory molecules. First we show that the Th2-like response to OVA/alum is dependent on B7.2 interaction with ligand since it can be abrogated by anti-B7.2 treatment. Expression of costimulatory molecules was then studied in mice immunized with OVA/alum in the absence or presence of B. abortus . B7.2, but not B7.1, was up-regulated on mouse non-T and T cells following immunization with B. abortus . Surprisingly, B. abortus induced down-regulation of CD28 and up-regulation of B7.2 on murine CD4 + and CD8 + T cells. These effects on T cells were maximal for CD28 and B7.2 at 40 to 48 h and were not dependent on interleukin-12 (IL-12) or IFN-γ. On the basis of these results, we propose that the IL-12/IFN-γ-independent inhibition of Th2 responses to OVA/alum is secondary to the effects of B. abortus on expression of costimulatory molecules on T cells. We suggest that down-regulation of CD28 following activation inhibits subsequent differentiation of Th0 into Th2 cells. In addition, decreased expression of CD28 and increased expression of B7.2 on T cells would favor B7.2 interaction with CTLA-4 on T cells, and this could provide a negative signal to developing Th2 cells.

Enhancement of immunity in the setting of HIV infection is difficult owing to loss of functional CD4+ T cells. The MHC class II-deficient mouse (II-/-) environment simulates that of the immunocompromised HIV-infected individual, since these mice have low CD4+ T cell numbers, defective CD4-dependent responses, and are susceptible to opportunistic infection. This strain was used to test whether heat-killed Brucella abortus (BA), covalently conjugated to the V3 peptide of HIV-1 (MN), could elicit anti-HIV responses. V3-BA, but not the T-dependent antigen V3-KLH, induced high levels of IL-12, IFN-gamma, and IL-10 mRNA in both wild-type (WT) and II-/- mice within 24 hr of injection. V3-BA-treated, but not V3-KLH-treated, II-/- mice developed serum IgG and IgA anti-V3 antibodies, with IgG2b and IgG3 as the predominant isotype. Viral neutralization studies, using a syncytium inhibition assay, demonstrated that the antibodies generated by V3-BA in II-/- mice were capable of neutralizing HIV. These experiments demonstrate that a heat-inactivated bacterium such as BA, when used as a carrier, can generate a cytokine environment that results in the production of neutralizing antiviral antibodies in an immunodeficient host. Such strategies could be important in the development of immunotherapies and vaccines for HIV-1 patients.

Development of a vaccine which is capable of generating a strong cellular immune response associated with gamma interferon (IFN-gamma) production and cytotoxic T-cell development requires that the immunogen be capable of inducing the secretion of interleukin-12 (IL-12), which is a pivotal factor for the differentiation of Th1 or Tc1 cells. We have previously shown that the heat-inactivated gram-negative bacterium Brucella abortus can induce IFN-gamma secretion by T cells. In the present study, we demonstrate that B. abortus and lipopolysaccharide (LPS) from B. abortus can induce IL-12 p40 mRNA expression and protein secretion by human elutriated monocytes (99% pure). p40 mRNA was detected within 4 h, and p40 protein could be measured at 24 h. This induction was abrogated by anti-CD14 monoclonal antibody, suggesting that monocytes recognize B. abortus via their receptor for LPS. The biological activity of IL-12 secreted by B. abortus-stimulated monocytes was demonstrated by its ability to upregulate IFN-gamma mRNA expression in T cells separated from monocytes and B. abortus by a transwell membrane. The B. abortus-induced IL-12 also enhanced NK cytolytic activity against K562 target cells. B. abortus was shown to rapidly increase the expression of the costimulatory molecules B7.1 and B7.2 and intercellular adhesion molecule 1 on human monocytes. Together, these data indicate that B. abortus can directly activate human monocytes and provide the cytokine milieu which would direct the immune response towards Th1-Tc1 differentiation.

We have previously shown that immunization of mice with human immunodeficiency virus (HIV)-derived proteins or peptides conjugated to inactivated Brucella abortus induces the secretion of virus-neutralizing antibodies, predominantly of the immunoglobulin G2a (IgG2a) isotype. In addition, B. abortus activates human CD4+ and CD8+ cells to secrete gamma interferon. Since these are both characteristics of a Th1-type immune response, which is associated with the development of cell-mediated immunity, it was important to determine if B. abortus conjugates would also act as a carrier to induce a cytotoxic T-lymphocyte (CTL) response. To test this hypothesis, we conjugated an 18-amino-acid peptide from the V3 loop of the MN strain of HIV-1 gp120 that contains both B- and cytotoxic T-cell epitopes to B. abortus (B. abortus-MN 18-mer). A 10-amino-acid fragment of this peptide has been shown to be the minimal CTL determinant presented by murine H-2Dd. It was found that two in vivo immunizations with 10(8) organisms of B. abortus-MN 18-mer followed by in vitro stimulation with peptide induced a virus-specific CTL response. Conjugation to B. abortus was required for in vivo priming, since there was no induction of memory CTLs when B. abortus was only mixed with peptide. Targets pulsed with peptide as well as those infected with a vaccinia virus encoding HIV gp160 were killed, demonstrating recognition of naturally processed envelope. Also, major histocompatibility complex-incompatible L cells which were infected with vaccinia viruses that encoded H-2Dd, but not H-2Kd, and pulsed with peptide were lysed. This demonstrated the appropriate major histocompatibility complex class I restriction. Treatment of the mice with anti-L3T4 prior to immunization caused a severe depletion of CD4+ lymphocytes, yet it did not decrease the CTL priming. Thus, inactivated B. abortus can induce non-CD4+ cells to produce the cytokines required for CTL induction. We conclude that B. abortus stimulates a cellular as well as a humoral immune response, even in the relative absence of CD4+ helper cells. It may be a particularly useful vaccine carrier in HIV-1-infected individuals or others with impaired CD4+ T-cell function.

Defining the pattern of lymphokine production associated with Brucella abortus is critical for advancing the development of B. abortus as a vaccine carrier. In the present study we investigated the ability of heat-inactivated B. abortus or lipopolysaccharide from B. abortus to induce lymphokine production from purified human T cells in vitro. Gamma interferon (IFN-gamma), interleukin-2 (IL-2), IL-4, and IL-5 induction was assayed by mRNA-specific PCR and by enzyme-linked immunosorbent assay and bioassay for protein production. Following depletion of monocytes and B cells, B. abortus increased IFN-gamma and IL-2 mRNA expression in purified T cells compared with expression in unstimulated cells. In contrast, no IL-5 mRNA expression and only transient low-level IL-4 mRNA expression and no IL-4 protein secretion were detected. Phytohemagglutinin or phorbol myristate acetate plus ionomycin induced mRNA and protein for all these cytokines. Similar results were obtained with LPS purified from B. abortus. Removal of NK cells did not reduce lymphokine production, and enriched NK cells did not express IFN-gamma mRNA or secrete IFN-gamma protein in response to B. abortus, indicating that NK cells were not the responding population. Both CD4+ and CD8+ populations produced IFN-gamma and IL-2 in response to B. abortus. Preincubation of resting T cells with B. abortus or LPS from B. abortus for 7 days induced their differentiation into Th1-like cells as judged by their subsequent lymphokine response to phorbol myristate acetate plus ionomycin. These results suggest that B. abortus can induce differentiation of Th0 into Th1-type cells.

Viral infections are often accompanied by pulmonary microvascular leakage and vascular endothelial dysfunction via mechanisms that are not completely defined. Here, we investigated the effect of the Toll-like receptor 3 (TLR3) ligand polyinosinic-polycytidylic acid [Poly(I:C)], a synthetic analog of viral double-stranded RNA (dsRNA) commonly used to simulate viral infections, on the barrier function and tight junction integrity of primary human lung microvascular endothelial cells. Poly(I:C) stimulated IL-6, IL-8, TNFα, and IFNβ production in conjunction with the activation of NF-κB and IRF3 confirming the Poly(I:C)-responsiveness of these cells. Poly(I:C) increased endothelial monolayer permeability with a corresponding dose- and time-dependent decrease in the expression of claudin-5, a transmembrane tight junction protein and reduction of CLDN5 mRNA levels. Immunofluorescence experiments revealed disappearance of membrane-associated claudin-5 and co-localization of cytoplasmic claudin-5 with lysosomal-associated membrane protein 1. Chloroquine and Bay11-7082, inhibitors of TLR3 and NF-κB signaling, respectively, protected against the loss of claudin-5. Together, these findings provide new insight on how dsRNA-activated signaling pathways may disrupt vascular endothelial function and contribute to vascular leakage pathologies.

Vaccine strategies need to take into account the balance of T helper subsets they induce. TH1 cells, which secrete IFN gamma and IL-2, are associated with CMI, rather than humoral responses, and afford protection against intracellular infections including parasites. In contrast, TH2 cells secrete IL-4, IL-5, and IL-10; elicit high-titer antibody responses and poor CMI; and are associated with susceptibility to infection with intracellular pathogens. Depending on the type of TH cell bias required, it is possible to manipulate the immune response to a protein or peptide by employing (1) different adjuvants, (2) conjugating the protein to various carriers, (3) immunizing in the presence of cytokines, (4) using alternative routes of administration, or (5) using different forms or doses of antigen. To apply these approaches to a particular vaccine, it is necessary to identify which component of the infectious agent (e.g., envelope protein or peptide) or allergen to target. Once the type of TH cell response that is protective is identified, it may be possible to combine a protein with an adjuvant or link it to a carrier that will promote responses towards the most advantageous TH subset.

It is known that the lipopolysaccharide (LPS) of gram-negative bacteria, in addition to being a potent adjuvant, is an effective carrier for covalently associated haptens. However, the toxic nature of most forms of LPS precludes their use as adjuvants or carriers for human vaccines. 4'-Monophosphoryl lipid A (MLA), a derivative of LPS with attenuated toxicity, is currently being tested in humans as an immunological adjuvant. In this study, MLA was tested for its ability to function as a carrier for a small hapten, the trinitrophenyl group (TNP). MLA was first modified by addition of 6-aminocaproic acid to the 6' position of the disaccharide backbone (Cap-MLA). TNP was then attached to Cap-MLA via the free amino group, yielding TNP-Cap-MLA. Immunization of normal mice with TNP-Cap-MLA resulted in high-titer anti-TNP responses of immunoglobulin M and all immunoglobulin G subclasses. Furthermore MLA, like other T-cell-independent type 1 (TI-1) carriers, induced responses in athymic and X-linked immunodeficient mice. In all cases, immunization with either MLA alone or TNP-Cap plus MLA failed to induce measurable anti-TNP antibodies of any isotype, indicating that covalent association of MLA and hapten was necessary for MLA's carrier activity to be manifested. These properties of MLA make it a potential candidate as a carrier for vaccine subunit components, such as small peptides, especially for situations in which T-cell help is impaired, as occurs following human immunodeficiency virus type 1 infection.

Therapeutic proteins are derived from complex expression/production systems, which can result in minor conformational changes due to preferential codon usage in different organisms, post-translational modifications, etc. Subtle conformational differences are often undetectable by bioanalytical methods but can sometimes profoundly impact the safety, efficacy and stability of products. Numerous bioanalytical methods exist to characterize the primary structure of proteins, post translational modifications; protein-substrate/protein/protein interactions and functional bioassays are available for most proteins that are developed as products. There are however few analytical techniques to detect changes in the tertiary structure of proteins suitable for use during drug development and quality control. For example, x-ray crystallography and NMR are impractical for routine use and do not capture the heterogeneity of the product. Conformation-sensitive antibodies can be used to map proteins. However the development of antibodies to represent sufficient epitopes can be challenging. Other limitations of antibodies include limited supply, high costs, heterogeneity and batch to batch variations in titer. Here we provide proof-of-principle that DNA aptamers to thrombin can be used as surrogate antibodies to characterize conformational changes. We show that aptamers can be used in assays using either an ELISA or a label-free platform to characterize different thrombin products. In addition we replicated a heat-treatment procedure that has previously been shown to not affect protein activity but can result in conformational changes that have serious adverse consequences. We demonstrate that a panel of aptamers (but not an antibody) can detect changes in the proteins even when specific activity is unaffected. Our results indicate a novel approach to monitor even small changes in the conformation of proteins which can be used in a routine drug-development and quality control setting. The technique can provide an early warning of structural changes during the manufacturing process that could have consequential outcomes downstream.

The design of vaccine strategies in general, and those for malaria in particular, need to take into account the balance of T helper subsets (TH) they induce. The TH1 cells, which secrete interferon-gamma and interleukin-2 (IL-2), are associated with cell-mediated immunity (CMI), rather than humoral responses, and afford protection against intracellular infections, including those caused by parasites. In contrast, the TH2 cells secrete IL-4, IL-5, and IL-10, elicit high titer antibody responses, provide poor CMI, and are often correlated with susceptibility to infection. Depending on the type of TH cell bias required, it is possible to manipulate the immune response to a protein/peptide by 1) using different adjuvants, 2) conjugating the protein to various carriers, 3) immunizing in the presence of cytokines, or 4) using alternative routes of administration. To apply these approaches to malarial vaccines, it is necessary to identify which stage(s) of the parasite to target and what type of TH cell bias is protective against that particular stage. We favor using carriers such as Brucella abortus, which focus the antigen on a specific particle and which can trigger a TH1 cell response.

In order to determine the carrier nature of lipopolysaccharide from Brucella abortus (LPS-BA) in evoking humoral responses, normal and immunodeficient mice were immunized with trinitrophenyl (TNP)-conjugated LPS-BA (TNP-LPS-BA) and the responses were compared with those to known T-dependent and T-independent antigens. TNP-LPS-BA, like T-independent type 1 (TI-1) antigens such as TNP-BA and TNP-LPS from Escherichia coli (TNP-LPS-EC), generated anti-TNP responses in BALB/c, athymic BALB/c nu/nu, and CBA/N mice. In contrast, N-2,4-dinitrophenyl-beta-alanylglycylglycyl-substituted keyhole limpet hemocyanin, a typical T-dependent antigen, was not immunogenic in athymic mice, and TNP-Ficoll (T-independent type 2) was ineffective in eliciting humoral responses in CBA/N mice. These results indicate that LPS from B. abortus acts as a TI-1 carrier in generating antibody responses. In C3H/HeJ mice, TNP-LPS-BA generated higher-titer immunoglobulin G1 (IgG1), IgG2a, and IgG2b anti-TNP antibodies than TNP-LPS-EC. Compared with those from BALB/c mice, pure resting B cells isolated from C3H/HeJ mice exhibited a 30-fold lower proliferative response to LPS-EC, whereas the LPS-BA response was reduced to a lesser extent (5-fold). This suggests that the disparity observed in antibody titers was due to different abilities of LPS from B. abortus and E. coli to stimulate C3H/HeJ B cells. The ability of LPS from B. abortus to act as a carrier in generating humoral immune responses indicates that LPS-BA can be substituted for whole B. abortus organisms in vaccine development.

The fusion of a protein with the Fc-region of an antibody is used to increase the circulating half-life of therapeutic proteins. Using a mouse hemophilia B (HB) model we compared the immune responses to infusions of recombinant human FIX (rFIX) and human FIX-mouse Fc (hFIX-mFc) fusion protein. HB mice received weekly IV injection of rFIX or hFIX-mFc for 5 weeks. Anti-FIX IgG, IgE and cytokines were measured. Splenic cells were used to determine FIX-specific T and B cell functional and memory responses. Our results showed that, following IV treatment with rFIX or hFIX-mFc, there are no statistically significant differences in the titers of anti-FIX IgG Ab and numbers of long term memory B cells. However, a significantly larger proportion of mice treated with rFIX developed elevated titers of total IgE which correlated with a Th2 bias following rFIX. In contrast, hFIX-mFc induced a Th1-like response and no detectable IgE. Our study suggests, that in mice, hFIX-mFc fusion protein promotes a Th1-like response and is less likely than rFIX to induce an IgE response, which may lead to anaphylaxis, a rare severe adverse event associated with the treatment of HB patients.

The inner-membrane protein BacA affects Brucella LPS structure. A bacA deletion mutant of Brucella abortus, known as KL7 (bacA(mut)-KL7), is attenuated in BALB/c mice and protects against challenge. Thus, bacA mutation was a candidate for incorporation into live attenuated vaccines. We assessed bacA(mut)-KL7 in 2 additional mouse strains: the more resistant C57BL/6 that produces interferon-gamma throughout the infection and the highly susceptible interferon-gamma-deficient C57BL/6 in which brucellae exhibit continual exponential growth. While it was hypothesized that bacA(mut)-KL7 would exhibit even greater attenuation relative to its parent strain B. abortus 2308 in C57BL/6 mice than it did in BALB/c mice, this was not the case. Moreover, it was more pathogenic in C57BL/6 interferon-gamma-deficient mice than 2308 causing abscesses and wasting even though the splenic loads of bacA(mut)-KL7 were significantly lower. These 2 observations were correlated, respectively, with an ability of IFNgamma-activated macrophages to equivalently control strains 2308 and bacA(mut)-KL7 and the ability of bacA(mut)-KL7 organism and its LPS to induce greater amounts of pro-inflammatory cytokines than 2308. We conclude that attenuation properties of bacA mutation are dependent upon the nature of the host but more importantly that bacterial gene deletion can result in increased host pathology without an increase in bacterial load, crucial considerations for vaccine design.

In this report we provide evidence, for the first time, that bacterial DNA in the context of heat-killed Brucella abortus (HKBA) engages TLR9 in dendritic cells (DC), resulting in a Th1-like cytokine response. This is based on the findings that HKBA induction of IL-12p40 is: 1) abolished in DC from TLR9−/− mice; 2) blocked by suppressive oligodeoxynucleotides; 3) simulated by bacterial DNA derived from HKBA; and 4) abrogated by DNase or methylation of the DNA from HKBA. Furthermore, the effect of HKBA can be inhibited by chloroquine, indicating that endosomal acidification is required and supporting the notion that DNA from HKBA is interacting with TLR9 at the level of the endosome, as is the case with CpG oligodeoxynucleotides. In addition to DC, HKBA can elicit IL-12p40 secretion from macrophages, in which case the effect is wholly MyD88 dependent but only partially TLR9 dependent. This probably explains why HKBA effects in vivo are only partially reduced in TLR9−/−, but absent in MyD88−/− mice. Because of their intimate interactions with T cells, the DC response is most likely to be critical for linking innate and adaptive immune responses, whereas the macrophage reaction may play a role in enhancing NK cell and bystander immune responses. In addition to IL-12p40, HKBA induces other Th1-like cytokines, namely, IFN-α and IFN-γ, in a TLR9-dependent manner. These cytokines are important in protection against viruses and bacteria, and their induction enhances HKBA as a potential carrier for vaccines.

Currently there is no effective treatment for inhalational anthrax beyond administration of antibiotics shortly after exposure. There is need for new, safe and effective treatments to supplement traditional antibiotic therapy. Our study was based on the premise that simultaneous inhibition of lethal toxin action with antibodies and blocking of bacterial growth by antibiotics will be beneficial for the treatment of anthrax. In this study, we tested the effects of a combination treatment using purified rabbit or sheep anti-protective antigen (PA) antibodies and the antibiotic ciprofloxacin in a rodent anthrax model. In mice infected with a dose of Bacillus anthracis Sterne strain corresponding to 10 LD(50), antibiotic treatment with ciprofloxacin alone only cured 50% of infected animals. Administration of anti-PA IgG in combination with ciprofloxacin produced 90-100% survival. These data indicate that a combination of antibiotic/immunoglobulin therapy is more effective than antibiotic treatment alone in a rodent anthrax model.

Cattle and humans are susceptible to infection with the Gram-negative intracellular bacterium Brucella abortus. Heat-killed B. abortus (HKBA) is a strong Th1 adjuvant and carrier. Previously, we have demonstrated that dendritic cells produce IL-12 in response to HKBA stimulation. In the present study, we use knockout mice and in vitro reconstitution assays to examine the contribution of signaling by Toll-like receptors (TLRs) and their immediate downstream signaling initiator, myeloid differentiation protein MyD88, in the activation following stimulation by HKBA. Our results show that HKBA-mediated induction of IL-12p40 and TNF is dependent on the adapter molecule MyD88. To identify the TLR involved in HKBA recognition, we examined HKBA responses in TLR2- and TLR4-deficient animals. TNF responses to HKBA were TLR4 independent; however, the response in TLR2-deficient mice was significantly delayed and reduced, although not completely abolished. Studies using Chinese hamster ovary/CD14 reporter cell lines stably transfected with either human TLR2 or human TLR4 confirmed the results seen with knockout mice, namely TLR2, but not TLR4, can mediate cellular activation by HKBA. In addition, human embryonic kidney 293 cells, which do not respond to HKBA, were made responsive by transfecting TLR2, but not TLR4 or TLR9. Taken together, our data demonstrate that MyD88-dependent pathways are crucial for activation by HKBA and that TLR2 plays a role in TNF, but not IL-12p40 pathways activated by this microbial product.

Passive antibody prophylaxis against human immunodeficiency virus type 1 (HIV-1) has been accomplished in primates, suggesting that this strategy may prove useful in humans. While antibody specificity is crucial for neutralization, other antibody characteristics, such as subclass, have not been explored. Our objective was to compare the efficiencies of immunoglobulin G (IgG) subclasses from polyclonal human HIV immune globulin (HIVIG) in the neutralization of HIV-1 strains differing in coreceptor tropism. IgG1, IgG2, and IgG3 were enriched from HIVIG by using protein A-Sepharose. All three subclasses bound major HIV-1 proteins, as shown by Western blot assay and enzyme-linked immunosorbent assay. In HIV-1 fusion assays using X4, R5, or X4R5 envelope-expressing effector cells, IgG3 more efficiently blocked fusion. In neutralization assays with cell-free viruses using X4 (LAI, IIIB), R5 (BaL), and X4R5 (DH123), a similar hierarchy of neutralization was found: IgG3 > IgG1 > IgG2. IgG3 has a longer, more flexible hinge region than the other subclasses. To test whether this is important, IgG1 and IgG3 were digested with pepsin to generate F(ab′)2fragments or with papain to generate Fab fragments. IgG3 F(ab′)2fragments were still more efficient in neutralization than F(ab′)2of IgG1. However, Fab fragments of IgG3 and IgG1 demonstrated equivalent neutralization capacities and the IgG3 advantage was lost. These results suggest that the IgG3 hinge region confers enhanced HIV-neutralizing ability. Enrichment and stabilization of IgG3 may therefore lead to improved HIVIG preparations. The results of this study have implications for the improvement of passive immunization with polyclonal or monoclonal antibodies and suggest that HIV-1 vaccines which induce high-titer IgG3 responses could be advantageous.

Brucella abortus is an intracellular pathogen that causes disease in cattle and in humans. The response against B. abortus involves the whole gamut of the immune system, from innate to adaptive immunity resulting from stimulation of antigen-presenting cells, NK cells, CD4(+) and CD8(+) T cells, and B cells.

CD4+ T cells play a central role in most effective immune responses to antigens and pathogens. Since the phenotype of CD4+ T cells induced defines the nature of the subsequent immune response, designing vaccines which favor the appropriate T cell subset is of critical importance. Two subsets of mature Th cells, Th1 and Th2, have been distinguished based upon the pattern of cytokines they produce (Mosmann and Coffman, 1989; Brown et al., 1989). Antigen-presenting cells (APC), which first contact and process antigens, present them to naive “Th0” cells in the context of certain cytokines and costimulatory signals. The microenvironment encountered by naive Th0 cells in conjuction with antigens influences their development towards a Thl or Th2 direction. In particular, IL-12, IFN-γ and IFN-α favor Th1 cell development, whereas IL-4 promotes differentiation into Th2 cells (Swain, 1993; Hsieh et al, 1993a; Seder et al., 1993; Brinkmann et al., 1993; Wenner et al., 1996). Differentiated, antigen-specific Thl cells produce IFN-γ, IL-2 and TNF-6 (Abbas et al., 1996). Th1 cells promote DTH and complement-fixing, IgG2a antibody production in mice, while the Th2 subset promotes the formation of IgG1 and allergy-mediating IgE antibodies in mice (Finkelman et al., 1990; Snapper and Paul, 1987; Finkelman et al., 1988b; Finkelman et al., 1988a; Finkelman et al, 1986). Evidence for Thl and Th2-like populations exists in human diseases such as leprosy, leishmaniasis, HIV (progression), atopy, and asthma (Secrist et al., 1993; Parronchi et al, 1991; Clerici and Shearer, 1993; Yamamura et al., 1991; Heinzel et al., 1991). Thl and Th2 subsets produce factors which enhance their own development and downregulate each other’s activity (Maggi et al., 1992; Powrie and Coffman, 1993; Schmitt et al., 1994; Swain et al, 1991; Tanaka et al., 1993).

Viral infections are often accompanied by pulmonary microvascular leakage and vascular endothelial dysfunction via mechanisms that are not completely defined. Here, we investigated the effect of the Toll-like receptor 3 ligand Poly (I:C), a synthetic analog of viral double-stranded RNA commonly used to simulate viral infections, on the barrier function and tight junction integrity of primary human lung microvascular cells. Poly (I:C) produced a dose- and time-dependent increase in endothelial monolayer permeability accompanied by a corresponding decrease in the expression of claudin-5, a transmembrane tight junction protein. Immunofluorescence experiments revealed disappearance of membrane-associated claudin-5 and co-localization of internalized claudin-5 with lysosomal-associated membrane protein 1 (LAMP-1), a marker of lysosomes, suggesting enhanced lysosomal degradation of claudin-5. Poly (I:C) also induced a dose- and time-dependent cleavage of TLR3 and TRIF as well as activation of NF-kB and IRF3, but not IRF7 (MyD88-dependent pathway), consistent with the direct stimulation of the TLR3 signaling. Pronounced inflammatory response with the release of IL-6, IL-8, MCP-1 and RANTES and a hallmark TLR3-mediated type 1 IFN production (IFNbeta) was also observed. Together, these findings provide new insight on how virally-activated signaling pathways may disrupt vascular endothelial function and contribute to vascular leakage pathologies.

Human immunodeficiency virus type 1 (HIV-1) infection is associated with loss of function and numbers of CD4+ T-helper cells. In order to bypass the requirement for CD4+ cells in antibody responses, we have utilized heat-inactivated Brucella abortus as a carrier. In this study we coupled a 14-mer V3 loop peptide (V3), which is homologous to 9 of 11 amino acids from the V3 loop of HIV-1 MN, and gp120 from HIV-1 SF2 to B. abortus [gp120(SF2)-B. abortus]. Our results showed that specific antibody responses, dominated by immunoglobulin G2a in BALB/c mice, were induced by these conjugates. Sera from the immunized mice bound native gp120 expressed on the surfaces of cells infected with a recombinant vaccinia virus gp160 vector (VPE16). Sera from mice immunized with gp120(SF2)-B. abortus inhibited binding of soluble CD4 to gp120, whereas sera from mice immunized with V3-B. abortus were ineffective. Sera from mice immunized with either conjugate were capable of blocking syncytium formation between CD4+ CEM cells and H9 cells chronically infected with the homologous virus. Sera from mice immunized with gp120(SF2)-B. abortus were more potent than sera from mice immunized with V3-B. abortus in inhibiting syncytia from heterologous HIV-1 laboratory strains. Importantly, in primary and secondary responses, V3-B. abortus evoked anti-HIV MN antibodies in mice depleted of CD4+ cells, and sera from these mice were able to inhibit syncytia. These findings indicate that B. abortus can provide carrier function for peptides and proteins from HIV-1 and suggest that they could be used for immunization of individuals with compromised CD4+ T-cell function.

Development of a vaccine to protect against infection with HIV-1 or to enhance antiviral immunity after HIV-1 has established itself in the host, is a major challenge to researchers. The virus has evolved many successful ways of evading the immune system which include its innate ability to mutate immunodominant regions and to lie dormant for many years as an integrated part of the human genome. In addition, the major target of HIV-1 is the CD4+ T-helper cell (Lane and Fauci, 1985). This cell plays a central role in the induction of effector cells responses to viruses. Early on following infection CD4+ Th cells are decreased in function and gradually are also decreased in number. Since these helper cell are required for specific immune responses to protein antigens, in terms of antibody and cytotoxic T cell responses (CTL), the host loses ability to attack the virus and to ward off other infections. The vaccines that are currently in clinical trials involve the use of proteins which probably require normal function of CD4+ Th in order that primary and secondary immune responses take place.

Brucella abortus may be useful as a component of vaccines. This is because it possesses several unique properties as a carrier that enable it to stimulate human B cells even in the relative absence of T cells. Human immunodeficiency virus type 1 proteins conjugated to B. abortus could induce neutralizing antibodies against human immunodeficiency virus type 1. Recently we showed that the characteristics of lipopolysaccharide (LPS) derived from B. abortus are similar to those of the whole bacterium in that the LPS acts as a T-independent type 1 carrier in mice. In this study we wanted to determine whether LPS derived from B. abortus is associated with the adverse effects seen with other bacterial endotoxins. LPS purified from B. abortus by butanol extraction was shown to have less than 2% (wt/wt) contamination by protein and less than 1% (wt/wt) contamination by nucleic acids and to contain 1% (wt/wt) ketodeoxyoctanic acid. Compared with LPS derived from Escherichia coli, B. abortus LPS was 10,000-fold less potent in eliciting fever in rabbits, 268-fold less potent in killing D-galactosamine-sensitized mice, and 1,400-fold and 400-fold less potent in inducing interleukin-1 beta and tumor necrosis factor alpha production, respectively. These results suggest that B. abortus LPS is much less likely than the LPS from E. coli to evoke endotoxic shock; therefore, it may be feasible to incorporate B. abortus as a component of vaccines.

Brucella abortus has been characterized as a T-independent type 1 antigen/carrier in human and murine antibody responses. In this report it is shown that BA can activate human CD3+ T cells to secrete interferon-gamma (IFN gamma). Unlike mitogens, such as phytohemagglutinin, this stimulation was associated with minimal T-cell proliferation or upregulation of interleukin-2 (IL-2) receptor. Monocytes inhibited BA-mediated IFN gamma secretion since their removal resulted in increased responses, whereas adding monocytes back to cultures caused inhibition. BA elicited IFN gamma from CD4+ and CD8+ T cells, although CD4+ T cells secrete significantly more (p less than 0.05) IFN gamma than CD8+ T cells. The ability of BA to elicit IFN gamma from human T cells was inhibited in the presence of anti-Tac, suggesting that BA also induces IL-2 secretion and that IL-2 is involved in BA-mediated IFN gamma secretion. Detectable IL-2 secretion was induced by BA in the presence of anti-Tac. Exogenous IL-2 acted synergistically with BA to enhance IFN gamma secretion, suggesting that the amount of IL-2 released by BA alone was insufficient for optimal IFN gamma release. Furthermore, addition of IL-2 to T cells from individuals with poor or absent responses to BA, including individuals infected with HIV-1, restored their ability to secrete IFN gamma in response to BA. These data indicate that BA is capable not only of activating human B cells but can also induce T cells, probably of the TH1 phenotype, to secrete IFN gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

In the present study inactivated human immunodeficiency virus type 1 (HIV-1) was conjugated to Brucella abortus and tested for immunogenicity in normal and anti-L3T4-treated BALB/c mice. HIV-BA was more immunogenic than uncoupled HIV in normal mice, since 6-fold less virus in HIV-BA preparations elicited higher titer responses than HIV-1 alone. Furthermore, the HIV-BA antibody response reached higher levels before the HIV-1 response. Immunoblot analysis showed that most of the HIV-1 antigens were recognized by antibodies induced by either HIV-1 or HIV-BA. Isotype analysis revealed that HIV-1 induced similar levels of IgG1 and IgG2a antibodies, whereas the IgG2a responses to HIV-BA were more pronounced than the IgG1 response. These different IgG subclass patterns suggest that conjugation of HIV-1 to BA changed the immunogenic nature of HIV-1. The requirement for helper T cells was examined by immunizing mice that were depleted of CD4+ T cells by in vivo anti-L3T4 treatment. Under these conditions the IgG responses to HIV-1 were completely eliminated. Although HIV-BA antibody responses were markedly reduced in anti-L3T4-treated mice, anti-HIV-1 antibodies, mainly of the IgG2a isotype, were produced. The antibodies generated by HIV-1 and HIV-BA immunization were also tested for their ability to inhibit syncytia formed by infecting CD4 + CEM cells with gp160 vaccinia. Sera from normal mice, immunized with either HIV-1 or HIV-BA were capable of inhibiting syncytia. In contrast, following anti-L3T4 treatment, only mice immunized with HIV-BA, but not HIV-1, produced antibodies capable of inhibiting syncytia.

We have previously reported the identification of highly conserved homologous regions located in the carboxy terminus of the HIV 1 gp41 (aa 837–844), and the amino-terminal of the beta chain of all human HLA class II antigens (aa 19–25). Murine monoclonal antibodies raised against synthetic peptides from these homologous regions bound not only to the isolated peptides, but also to “native” HLA class II molecules on cells. Screening of sera from HIV 1 infected individuals revealed high frequency of sera (35%) containing anti-class II crossreactive antibodies (CRAb), not only in AIDS patients, but also in early, asymptomatic patients. The CRAb containing sera caused potent inhibition of normal CD4-bearing cells’ proliferative responses to tetanus toxoid in vitro. They could also kill class II bearing cells by ADCC. The possible contribution of these antibodies to the establishment of immunodeficiency state in HIV 1 infected individuals and/or to disease progression, was examined in two clinical studies: I. Asymptomatic patients were tested in parallel for their PBL responses to flu/tetanus, HLA alloantigens, and PHA (proliferation and IL2 production), and for the presence of anti-class II CRAb. About 50% of these patients showed a selective loss of their in vitro responses to recall antigens (flu/tetanus), which depend on CD4+ cells, while still responding to PHA and ALLO. Interestingly, positive correlation was found (P>0.001) between patients’ lack of responsiveness to flu/tetanus and the presence in their sera of anti-class II CRAb. II. Retrospective study of HIV 1-infected hemophiliacs, suggest that patients with high titers of CRAb early in the disease progressed faster to full blown disease.