Development Of Antibodies In Mice Research Articles

Anti-FVIII Antibody and Inhibitor Development Patterns in Hemophilia A Mouse Models: A Systematic Review

Background: Management of hemophilia A with exogenous factor VIII (FVIII) infusions can result in the development of alloantibodies that neutralize FVIII activity (inhibitors). The presence of FVIII inhibitors is associated with high morbidity and a decreased quality of life. Hemophilia A mouse models are a valuable tool for studying inhibitor development and hemophilia A. These models have been shown to develop inhibitor upon treatment with human FVIII. However, there are numerous hemophilia A mouse models, each with potentially varying patterns of inhibitor development. Objective: To identify existing hemophilia A mouse models and characterize anti-FVIII antibody and inhibitor development patterns. Methods: A systematic literature review was conducted using Ovid MEDLINE and EMBASE. The Databases were searched from inception through May 2019 for full-length publications, published abstracts, and conference proceedings that reported use of mouse models of hemophilia A to investigate total anti-FVIII antibody levels and/or inhibitor titers. The search strategy used the categorical search terms: "hemophilia A" AND "mouse" AND "animal model" AND "inhibitor" (Figure 1). Results: Our review consisted of a total of 88 studies which reported the use of 11 different hemophilia A mouse models. Out of the 88 studies, 19 studies only reported total anti-FVIII antibody data without reporting inhibitor data. In 30% of the studies that reported both total anti-FVIII antibody and inhibitor data, not all mice that developed anti-FVIII antibodies had positive inhibitor titres. Our systematic review identified the E16 - C57BL/6 model as the most frequently used model, as 27 studies (31%) reported using this model. The E16 - C57BL/6, E16 - Balb/c, E16 - C57BL6/129, E17 - C57BL/6, E17 - C57BL6/129, and FVIII total knockout (TKO) models demonstrate high immunogenicity to exogenous FVIII and were used in 65% of the studies. These mice all consistently develop anti-FVIII antibodies and inhibitors at >95% incidence following treatment with at least 4 doses of full-length recombinant human FVIII (rhFVIII). The E16 - R593C model and the E17 - HLA-DRB1*1501 model both display anti-FVIII antibody and inhibitor development patterns that are representative of the human clinical scenario (~30% inhibitor development incidence). The only model completely immunotolerant to rhFVIII is the E17 - subline E model generated by van Helden et al. (2011) which is a transgenic mouse line on the hemophilic E17 background that expresses human full-length F8 cDNA. Treatment with 8 weekly doses of native human FVIII resulted in no anti-FVIII antibody or inhibitor development in this model. Conclusions: This systematic review demonstrated that mice of the most frequently used models almost always develop inhibitors when treated with full-length rhFVIII. Humanized models may be ideal for use in studies that aim to replicate human inhibitor development patterns. In addition, immunotolerant models may be best used for in vivo studies in hemophilia A that aim to avoid both antibody and inhibitor development. Lastly, this systematic review emphasises that it is important to report both anti-FVIII antibodies and neutralizing activity in Bethesda Units since in 30% of the studies that reported both values, the incidence of inhibitor development was less than the incidence of anti-FVIII antibody development in mice. Disclosures Matino: Bioviiix: Honoraria; Roche: Research Funding; Sanofi: Honoraria; Sobi: Honoraria, Research Funding; Bayer: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding.

Role of Protein A in the Evasion of Host Adaptive Immune Responses by Staphylococcus aureus

ABSTRACTHeritable defects in human B cell/antibody development are not associated with increased susceptibility to Staphylococcus aureus infection. Protein A (SpA), a surface molecule of S. aureus, binds the Fcγ domain of immunoglobulin (Ig) and cross-links the Fab domain of VH3-type B cell receptors (IgM). Here we generated S. aureus spa variants harboring amino acid substitutions at four key residues in each of the five Ig-binding domains of SpA. Wild-type S. aureus required SpA binding to Ig to resist phagocytosis and SpA-mediated B cell receptor cross-linking to block antibody development in mice. The spaKKAA mutant, which cannot bind Ig or IgM, was phagocytosed and elicited B cell responses to key virulence antigens that protected animals against lethal S. aureus challenge. The immune evasive attributes of S. aureus SpA were abolished in µMT mice lacking mature B cells and antibodies. Thus, while wild-type S. aureus escapes host immune surveillance, the spaKKAA variant elicits adaptive responses that protect against recurrent infection.

Evaluation of Adjuvant Regimens for Antibody Development in Mice

To obtain antibodies suitable for in vitro diagnostic immunoassays, animals must be immunized with an adjuvant that will elicit a high titer, high affinity response. We have previously shown, in rabbits, that Adjulite® Freund's adjuvant is successful in developing this immune response. To continue our studies, Adjulite® Freund's and MPL+TDM adjuvants were assessed for their ability to generate a high affinity immune response in mice. Two discrete, ~20 kDa, proteins were used to immunize CAF1/J and/or RBF/DnJ mice. The antigens were emulsified in either Adjulite® Freund's adjuvant or MPL+TDM adjuvant. Sera samples taken following the second immunization were evaluated for antibody titer and relative affinity in a microtiter enzyme immunoassay by testing for antibody reactivity to limiting amounts of the administered antigen. Results showed that, while both adjuvants were capable of yielding a similar relative affinity, animals immunized with Adjulite® Freund's adjuvant yielded a statistically significant higher titer in both antigen studies. In addition, in the first study, CAF1/J mice yielded a higher relative affinity immune response compared to RBF/DnJ mice with the average titer between the mouse strains remaining similar. In conclusion, these studies demonstrated that Adjulite® Freund's adjuvant generated both a high affinity and a high titer immune response for two different ~20kDa proteins in mice.

Immunoregulatory Mechanisms of PF4/Heparin Antibody Production In Vivo.

Heparin-Induced Thrombocytopenia (HIT) is a prothrombotic disorder caused by antibodies directed to complexes of platelet factor 4 (PF4) and heparin. Little is known about the immune basis of HIT, such as why serologic responses develop commonly in certain clinical settings or why immune responses may be transient in humans. To gain insight into the immune mechanisms governing PF4/heparin antibody production in vivo, we initiated studies in a recently developed murine immunization model of HIT, in which mice are injected with PF4/heparin antigen by retro-orbital injection daily for five days without adjuvant. PF4/heparin antibody development in mice is monitored weekly by a murine PF4/heparin (mP+H) ELISA. Immunizations of identical doses of PF4/heparin antigen in BALB/c and C57BL/6 (C57) mice resulted in marked strain-dependent differences in immune susceptibility, temporal kinetics and secondary recall. BALB/c mice showed more variable sensitization to mP+H injections (See Figure below) and manifested lower peak antibody levels (mean peak A450nm± SD: 0.153±0.1) than C57 mice (0.91±0.73). Time course of antibody formation was also strain-dependent. Peak antibody levels occurred ~ 45 days after start of immunization in BALB/c mice as compared to 15 days in C57 mice. Antigen rechallenge with one dose of antigen 65–85 days after primary sensitization revealed presence of variable immune recall in BALB/c mice (increased antibody levels in 3/7 mice) as compared to uniform recall in C57 mice (5/5 mice). We then initiated studies to investigate the immunoregulatory mechanisms underlying strain-dependent responses to mP+H antigen. Because isotype switching and expression of IgG subclasses is influenced by T helper (Th) cell subsets, we examined IgG subclasses in BALB/c and C57 mice as a surrogate marker of Th cytokine expression. C57 mice showed IgG2b>IgG1 anti-mP+H indicative of Th1 cytokine response, while BALB/c mice showed an IgG1>IgG2a response, a Th2 predominant response. Taken together, these findings of strain-dependent differences to mP+H indicate that variable expression of human HIT may be similarly governed by differences in peripheral immunoregulatory mechanisms. [Display omitted]

Mapping of genes that control the antibody response to human factor IX in mice

AbstractWe tested the hypothesis that the antibody response to human factor IX in mice is controlled by genetic factors, especially histocompatibility antigens. Seven inbred mouse strains were immunized against human factor IX by adenoviral gene transfer or serial injections of human factor IX protein. A/J mice had the highest antibody response and 2 C57 mouse strains had the lowest response. We used the adenovirus vector to immunize 26 recombinant inbred mouse strains (AXB and BXA) derived from A/J and C57BL/6J mice and observed highly significant linkage (logarithmic odds [LOD] scores ∼4.8) for the polymorphic D17Mit62 marker that is 1 centimorgan (∼300 000 base pair [bp]) from the mouse major histocompatibility complex (MHC) locus (H-2). Experiments in mice with chimeric MHC genes indicated that class IaK or class II H-2 (or both) genes were critical, but other genes contributed to the antibody response. Polymorphic markers from chromosomes 1 and 10 that are near important immunoregulatory genes such as interleukin 10 and the interferon-γ gene show suggestive linkage (LOD scores of ∼2.3-2.6) to the factor IX antibody response. This study confirms the hypothesis that H-2 (and other) genes control factor IX antibody development in mice and suggests their potential importance for factor IX antibody development in humans with hemophilia B.

Development and persistence of antibodies to Orientia tsutsugamushi in the roof rat, Rattus rattus and laboratory mice following attachment of naturally infected Leptotrombidium deliense

The development and persistence of antibodies to Orientia tsutsugamushi in Rattus rattus and laboratory mice following infection from the bite of naturally infected Leptotrombidium deliense is reported. Antibodies in R. rattus were first detected using an indirect immunoperoxidase method 2 weeks after attachment of an infected mite. The IgM antibody response was mild, and was detected 2, 4 and 6 weeks after infection, while the IgG response was stronger and was detected from 2 to 19 weeks after infection, when the tests ceased. Active rickettsiae were isolated from R. rattus 1–8 weeks after attachment of infected L. deliense, but rats were not adversely affected by infection and appeared to behave in the same way as uninfected rats. In contrast to rats, laboratory mice were killed by O. tsutsugamushi following infection by attachment of infected L. deliense. The development of antibodies in mice was not detected before gross symptoms of infection were observed. Antibodies were first detected 14 days after attachment of infected mites, when mice were close to death.