The prevalence of HPA-1a alloimmunization and the potential risk of FNAIT depend on both the DRB3*01:01 allele and associated DR-DQ haplotypes.

Alloimmunization against human platelet antigen (HPA)-1a during pregnancy can cause foetal/neonatal alloimmune thrombocytopenia (FNAIT) and severe bleeding in the foetus or newborn and likely depends on several factors. HPA-1a alloimmunization is associated with DRB3*01:01, which is associated with several DR-DQ haplotypes. However, it is not known to what extent these haplotypes contribute to the prevalence of HPA-1a alloimmunization. HPA-1a-alloimmunized women, identified in a prospective study, and random donors were typed for selected DRB3, DRB4, DRB1, DQA1 and DQB1 alleles to determine allele and DR-DQ haplotype frequencies. DRB3*01:01 was carried by 94% HPA-1a-immunized women compared to 27% in the general population. In the first population, the DR3-DQ2 haplotype was overrepresented (P < .003). The prevalence of HPA-1a alloimmunization was estimated to be about twice as frequent with DR3-DQ2 compared to DR13-DQ6, together accounting for about 90% of DRB3*01:01-positive individuals. Further, we examined DQB1*02 and DRB4*01:01 alleles for their reported association with HPA-1a alloimmunization, in the context of DR-DQ haplotypes. Since ~ 80% of DQB1*02 alleles are linked to the DR3-DQ2 haplotype, the association might be coincidental. However, the DQB1*02:02-associated DR7-DQ2 haplotype was also overrepresented in alloimmunized women, suggesting a role for this allele or haplotype in HPA-1a alloimmunization. As DRB4*01:01 is predominantly associated with the DR7-DQ2 haplotype in HPA-1a-alloimmunized individuals, the reported association with FNAIT may be coincidental. Typing for DR-DQ haplotypes revealed important genetic associations with HPA-1a alloimmunization not evident from typing individual alleles, and the presence of different DRB3-associated DR-DQ haplotypes showed different prevalence of HPA-1a alloimmunization.

A combined effect of anti-HPA-1a and anti-HLA Class I in pregnancy?

BACKGROUND: Maternal anti-human leukocyte antigen (HLA) Class I is commonly detected alongside anti-human platelet antigen (HPA)-1a in fetal and neonatal alloimmune thrombocytopenia (FNAIT). Little is known regarding whether the presence of anti-HLA Class I may exert an additive effect on the risk and severity of FNAIT. METHODS AND MATERIALS: We reanalyzed samples originally collected as part of a large Norwegian screening study on FNAIT during 1995-2004. This study identified and managed 170 pregnancies where the mother was HPA-1a negative and had detectable anti-HPA-1a during pregnancy. Maternal samples from 166 of these pregnancies were rescreened for anti-HLA Class I, revealing 111 (67%) that were antibody positive. Various regression models were used to assess if and how maternal anti-HLA Class I influenced the neonatal platelet count. RESULTS AND CONCLUSIONS: Unadjusted neonatal platelet counts and the frequency of neonatal thrombocytopenia was not significantly affected by the presence of anti-HLA Class I alongside anti-HPA-1a, but results from regression analyses revealed a possible increased risk when the mother was nulliparous. These results warrant further investigation.

Strategies to develop a prophylaxis for the prevention of HPA-1a immunization and fetal and neonatal alloimmune thrombocytopenia.

Anti-HPA-1a-antibodies are the main cause of fetal and neonatal alloimmune thrombocytopenia (FNAIT) which may result in intracranial hemorrhage (ICH) and death among fetuses and newborns. Advances in understanding the pathogenesis of FNAIT and proof of concept for prophylaxis to prevent immunization suggest that development of hyperimmune anti-HPA-1a IgG aimed at preventing immunization against HPA-1a and FNAIT is feasible. Anti-HPA-1a IgG can be obtained either by isolating immunoglobulin from already-immunized women or by development of monoclonal anti-HPA-1a antibodies. Here we discuss recent advances that may lead to the development of a prenatal and postnatal prophylactic treatment for the prevention of HPA-1a-associated FNAIT and life-threatening FNAIT-induced complications.

Anti-human platelet antigen (HPA)-1a antibodies may affect trophoblast functions crucial for placental development: a laboratory study using an in vitro model.

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a bleeding disorder caused by maternal antibodies against paternal human platelet antigens (HPAs) on fetal platelets. Antibodies against HPA-1a are accountable for the majority of FNAIT cases. We have previously shown that high levels of maternal anti-HPA-1a antibodies are associated with clinically significant reduced birth weight in newborn boys. Chronic inflammatory placental lesions are associated with increased risk of reduced birth weight and have previously been reported in connection with FNAIT pregnancies. The HPA-1a epitope is located on integrin ß3 that is associated with integrin αIIb (the fibrinogen receptor) on platelets and megakaryocytes. Integrin ß3 is also associated with integrin αV forming the αVß3 integrin heterodimer, the vitronectin receptor, which is expressed on various cell types, including trophoblast cells. It is therefore thinkable that maternal anti-HPA-1a antibodies present during early pregnancy may affect placenta function through binding to the HPA-1a antigen epitope on invasive throphoblasts. The aim of the study was to examine whether interaction of a human anti-HPA-1a monoclonal antibody (mAb) with HPA-1a on trophoblast cells affect adhesion, migration and invasion of extravillous trophoblast cells. METHODS: An in vitro model with human anti-HPA-1a mAb, clone 26.4, and the first trimester extravillous trophoblast cell line HTR8/SVneo was employed. The xCELLigence system was utilized to assess the possible effect of anti-HPA-1a mAb on adhesion and migration of HTR8/SVneo cells. Specially designed chambers precoated with Matrigel were used to assess the effect on the invasive capacity of cells. RESULTS: We found that human anti-HPA-1a mAb 26.4 partially inhibits adhesion and migratory capacity of HTR8/SVneo cells. CONCLUSIONS: Our findings suggest that anti-HPA-1a antibodies may affect trophoblast functions crucial for normal placental development. Future studies including primary throphoblast cells and polyclonal anti-HPA-1a antibodies are needed to confirm these results.

Characterization of a human platelet antigen-1a-specific monoclonal antibody derived from a B cell from a woman alloimmunized in pregnancy.

Human platelet Ag (HPA)-1a, located on integrin ß3, is the main target for alloantibodies responsible for fetal and neonatal alloimmune thrombocytopenia (FNAIT) in the white population. There are ongoing efforts to develop an Ab prophylaxis and therapy to prevent or treat FNAIT. In this study, an mAb specific for HPA-1a, named 26.4, was derived from an immortalized B cell from an alloimmunized woman who had an infant affected by FNAIT. It is the only HPA-1a-specific human mAb with naturally paired H and L chains. Specific binding of mAb 26.4, both native and recombinant forms, to platelets and to purified integrins αIIbß3 (from platelets) and αVß3 (from trophoblasts) from HPA-1a(+) donors was demonstrated by flow cytometry and surface plasmon resonance technology, respectively. No binding to HPA-1a(-) platelets or integrins was detected. Moreover, the Ab binds with higher affinity to integrin αVß3 compared with a second HPA-1a-specific human mAb, B2G1. Further in vitro experimentation demonstrated that mAb 26.4 can opsonize HPA-1a(+) platelets for enhanced phagocytosis by monocytes, inhibit binding of maternal polyclonal anti-HPA-1a Abs, and weakly inhibit aggregation of HPA-1a-heterozygous platelets, the latter with no predicted clinical relevance. Thus, mAb 26.4 is highly specific for HPA-1a and could potentially be explored for use as a prophylactic or therapeutic reagent for FNAIT intervention and as a phenotyping reagent to identify women at risk for immunization.

A prominent lack of IgG1-Fc fucosylation of platelet alloantibodies in pregnancy.

Immunoglobulin G (IgG) formed during pregnancy against human platelet antigens (HPAs) of the fetus mediates fetal or neonatal alloimmune thrombocytopenia (FNAIT). Because antibody titer or isotype does not strictly correlate with disease severity, we investigated by mass spectrometry variations in the glycosylation at Asn297 in the IgG Fc because the composition of this glycan can be highly variable, affecting binding to phagocyte IgG-Fc receptors (FcγR). We found markedly decreased levels of core fucosylation of anti-HPA-1a-specific IgG1 from FNAIT patients (n = 48), but not in total serum IgG1. Antibodies with a low amount of fucose displayed higher binding affinity to FcγRIIIa and FcγRIIIb, but not to FcγRIIa, compared with antibodies with a high amount of Fc fucose. Consequently, these antibodies with a low amount of Fc fucose showed enhanced phagocytosis of platelets using FcγRIIIb(+) polymorphonuclear cells or FcγRIIIa(+) monocytes as effector cells, but not with FcγRIIIa(-) monocytes. In addition, the degree of anti-HPA-1a fucosylation correlated positively with the neonatal platelet counts in FNAIT, and negatively to the clinical disease severity. In contrast to the FNAIT patients, no changes in core fucosylation were observed for anti-HLA antibodies in refractory thrombocytopenia (post platelet transfusion), indicating that the level of fucosylation may be antigen dependent and/or related to the immune milieu defined by pregnancy.

Towards a prophylactic treatment of HPA-related foetal and neonatal alloimmune thrombocytopenia.

PURPOSE OF REVIEW: The purpose of the review is to show the similarities between haemolytic disease of the foetus and newborn (HDFN) and foetal and neonatal alloimmune thrombocytopenia (FNAIT) and to describe the background and challenges related to the current endeavours of developing a prophylaxis against FNAIT. The rationale for this prophylaxis is similar to the prophylaxis which has been used with great success for the last 40 years against RhD-associated HDFN. The idea is to prevent human platelet antigen (HPA)-1a-associated FNAIT by administering anti-HPA-1a immunoglobulin G (IgG) to nonimmunized HPA-1a-negative women after delivery of an HPA-1a-positive child. RECENT FINDINGS: Results from a Norwegian screening and intervention study on FNAIT have indicated that about 75% of women with antibodies against HPA-1a are immunized in relation to delivery. This observation leads to the possibility of preventing HPA-1a-associated FNAIT in the same way as today's prevention of HDFN. Results from a proof-of-concept study in a murine FNAIT model have shown that the production of alloantibodies against platelets can be suppressed by administrating antiplatelet antibodies after the antigenic challenge. Even more interesting, the prophylactic antiplatelet antibodies could also significantly reduce the clinical consequences of FNAIT in this FNAIT model. SUMMARY: These novel observations have paved the way for clinical studies. Production and testing of anti-HPA-1a IgG for clinical use will be carried out by a European Union-funded consortium. If the results from the clinical trial are favourable, there is a chance that a medicinal product for the prevention of FNAIT will be available within this decade.

An HPA-1a-positive platelet-depleting agent for prevention of fetal and neonatal alloimmune thrombocytopenia: a randomized, single-blind, placebo-controlled, single-center, phase 1/2 proof-of-concept study.

BACKGROUND: Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a rare and potentially life-threatening bleeding disorder of the fetus/newborn. Antibodies against human platelet antigen 1a (HPA-1a) are associated with the most frequent FNAIT cases. There are no approved therapies for FNAIT prevention or treatment. RLYB211 is a polyclonal HPA-1a hyperimmune IgG being developed to prevent FNAIT. OBJECTIVES: To investigate whether a single dose of anti-HPA-1a (1000 IU) could markedly accelerate the elimination of HPA-1ab platelets transfused into healthy, HPA-1a-negative participants as compared with placebo. METHODS: This randomized, single-blind, placebo-controlled, single-center, phase 1/2 proof-of-concept study (EudraCT: 2019-003459-12) included HPA-1a- and HLA-A2-negative healthy men. Cohort 1 received intravenous RLYB211 or placebo 1 hour after transfusion of HPA-1ab platelets. Cohort 1B received RLYB211 or placebo, followed by platelet transfusion 1 week later. Primary endpoint was the half-life of transfused platelets in circulation after administration of RLYB211 or placebo, determined by flow cytometry. Proof of concept was ≥90% reduction of half-life relative to placebo. RESULTS: Twelve participants were allocated to cohort 1 or 1B and randomized to receive RLYB211 (n = 9) or placebo (n = 3). RLYB211 markedly accelerated the elimination of HPA-1ab platelets in all participants vs placebo. In cohort 1B, this effect was observed 7 days after RLYB211 administration. Two treatment-emergent adverse events were possibly related to treatment, both in RLYB211-treated participants. No participants developed HPA-1a antibodies at 12 or 24 weeks. CONCLUSION: These data support the hypothesis that anti-HPA-1a could be used as prophylaxis in women at risk of having an FNAIT-affected pregnancy.

Toward a prophylaxis against fetal and neonatal alloimmune thrombocytopenia: induction of antibody-mediated immune suppression and prevention of severe clinical complications in a murine model.

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a severe bleeding disorder caused by maternal antibody-mediated destruction of fetal or neonatal platelets (PLTs). Results from our recent large screening study suggest that the pathophysiology of FNAIT is more similar to hemolytic disease of the fetus and newborn (HDFN) than previously thought. Immunization against HPA-1a might therefore be preventable by a prophylactic regimen of inducing antibody-mediated immune suppression (AMIS), which has been documented to be a useful prophylaxis against HDFN. This preclinical proof-of-concept study investigated whether passive administration of anti-ß3 integrin could induce AMIS and thereby prevent clinical complications of FNAIT. STUDY DESIGN AND METHODS: A murine model of FNAIT using ß3 integrin (GPIIIa)-deficient (ß3-/-) mice was employed for this study. AMIS in ß3-/- mice was induced by intravenous administration of human anti-HPA-1a immunoglobulin G or murine anti-ß3 antisera given as prophylaxis after transfusion of HPA-1a-positive human PLTs or murine wild-type PLTs, respectively. RESULTS: AMIS against both human and murine PLT antigens was induced using this prophylactic approach, reducing the amount of maternal PLT antibodies by up to 90%. Neonatal PLT counts were significantly increased and pregnancy outcome was improved in a dose-dependent manner. The incidence of intracranial hemorrhage, miscarriage, and dead-born pups in mice receiving high-dose prophylaxis was reduced to that of normal controls. We also observed that the severity of thrombocytopenia inversely correlated with birth weight. CONCLUSION: This work conceptually proves that prophylactic administration of PLT antibodies induces AMIS and prevents poor pregnancy outcome in FNAIT.

The development of severe neonatal alloimmune thrombocytopenia due to anti-HPA-1a antibodies is correlated to maternal ABO genotypes.

BACKGROUND: Maternal alloantibodies against HPA-1a can cross placenta, opsonize foetal platelets, and induce neonatal alloimmune thrombocytopenia (NAIT). In a study of 100, 448 pregnant women in Norway during 1995-2004, 10.6% of HPA-1a negative women had detectable anti-HPA-1a antibodies. DESIGN AND METHODS: A possible correlation between the maternal ABO blood group phenotype, or underlying genotype, and severe thrombocytopenia in the newborn was investigated. RESULTS: We observed that immunized women with blood group O had a lower risk of having a child with severe NAIT than women with group A; 20% with blood group O gave birth to children with severe NAIT, compared to 47% among the blood group A mothers (relative risk 0.43; 95% CI 0.25-0.75). CONCLUSION: The risk of severe neonatal alloimmune thrombocytopenia due to anti-HPA-1a antibodies is correlated to maternal ABO types, and this study indicates that the observation is due to genetic properties on the maternal side.

Platelet antibodies and fetal growth: maternal antibodies against fetal platelet antigen 1a are strongly associated with reduced birthweight in boys.

OBJECTIVE: To assess whether maternal HPA 1a alloimmunization is associated with birthweight. DESIGN: A retrospective observational cohort study. SETTING: The national reference laboratory for clinical platelet immunology at a university hospital. POPULATION: 165 HPA 1a incompatible pregnancies identified from a recent screening study of 100 448 women (124 pregnancies) and the national reference laboratory for clinical platelet immunology (41 pregnancies). METHODS: A linear mixed model analysis was used to assess whether maternal anti-HPA 1a antibodies were associated with birthweight. A generalized linear model was used to assess maternal anti-HPA 1a antibodies as risk factor for small-for-gestational age neonates. Both models were adjusted for gestational age at time of delivery, maternal age, parity, smoking habits during pregnancy, preeclampsia, diabetes mellitus and fetal sex. MAIN OUTCOME MEASURES. Maternal anti-HPA 1a antibody as risk factor of reduced birthweight and small-for-gestational age neonates. RESULTS: The level of maternal anti-HPA 1a antibodies was significantly associated with birthweight and risk of small-for-gestational age neonates after correcting for confounding variables (p<0.001). However, this association was only significant for boys. When the mother had high levels of anti-HPA 1a antibodies during pregnancy, the adjusted mean birthweight in boys was 530g lower compared with anti-HPA 1a antibody negative pregnancies (p<0.001). CONCLUSIONS: A linear relation between maternal anti-HPA 1a antibody levels and reduced birthweight in boys was demonstrated. Reduced birthweight should be considered a possible complication of fetal and neonatal alloimmune thrombocytopenia.

Prevention of Fetal/Neonatal Alloimmune Thrombocytopenia in Mice: Biochemical and Cell Biological Characterization of Isoforms of a Human Monoclonal Antibody.

Maternal alloantibodies toward paternally inherited Ags on fetal platelets can cause thrombocytopenia and bleeding complications in the fetus or neonate, referred to as fetal and neonatal alloimmune thrombocytopenia (FNAIT). This is most commonly caused by Abs against the human platelet Ag (HPA)-1a in Caucasians, and a prophylactic regimen to reduce the risk for alloimmunization to women at risk would be beneficial. We therefore aimed to examine the prophylactic potential of a fully human anti-HPA-1a IgG1 (mAb 26.4) with modified Fc region or altered N-glycan structures. The mAb 26.4 wild-type (WT) variants all showed efficient platelet clearance capacity and ability to mediate phagocytosis independent of their N-glycan structure, compared with an effector silent variant (26.4.AAAG), although the modified N-glycan variants showed differential binding to FcγRs measured in vitro. In an in vivo model, female mice were transfused with platelets from transgenic mice harboring an engineered integrin ß3 containing the HPA-1a epitope. When these preimmunized mice were bred with transgenic males, Abs against the introduced epitope induced thrombocytopenia in the offspring, mimicking FNAIT. Prophylactic administration of the mAb 26.4.WT, and to some extent the mAb 26.4.AAAG, prior to platelet transfusion resulted in reduced alloimmunization in challenged mice and normal platelet counts in neonates. The notion that the effector silent variant hampered alloimmunization demonstrates that rapid platelet clearance, as seen with mAb 26.4.WT, is not the sole mechanism in action. Our data thus successfully demonstrate efficient Ab-mediated immunosuppression and prevention of FNAIT by anti-HPA-1a monoclonal variants, providing support for potential use in humans.

T-cell responses associated with neonatal alloimmune thrombocytopenia: isolation of HPA-1a-specific, HLA-DRB3*0101-restricted CD4+ T cells.

T-cell responses have been implicated in the development of HPA-1a-induced neonatal alloimmune thrombocytopenia (NAIT). However, HPA-1a-specific T cells have neither been isolated nor characterized. Here, we aimed to determine whether HPA-1a-specific T cells could be isolated from HPA-1a-immunized women. In the present study, peripheral blood mononuclear cells (PBMCs) from an HPA-1a-alloimmunized woman were cultured for weeks in the presence of HPA-1a peptide, labeled with CFSE, and assayed for antigen-specific proliferation. Individual proliferating cells were isolated by fluorescence-activated cell sorting and expanded in culture. Antigen specificity and HLA restriction were determined by cytokine secretion (enzyme-linked immunospot [ELISPOT]) and proliferation assays. Several CD3(+)CD4(+) T-cell clones were isolated that proliferated and secreted cytokines in response to HPA-1a peptide. Two of these clones have been established in long-term culture in our laboratory. Both of these recognize synthetic as well as naturally processed HPA-1a antigen, and the recognition is restricted by the MHC molecule HLA-DRB3*0101 that is strongly associated with NAIT. These HPA-1a-specific T-cell clones represent unambiguous evidence for the association of T-cell responses with NAIT, and they will serve as unique tools to elucidate the cellular immune response that may result in NAIT.

The cellular immunobiology associated with fetal and neonatal alloimmune thrombocytopenia.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by maternal antibodies that cross the placenta in connection with pregnancy and destroy fetal platelets. Recently, maternal T cell responses associated with FNAIT have been studied at the clonal level. These T cell clones recognize an integrin ß3 epitope, which is anchored to the HLA-DRB3∗0101-encoded MHC molecule DR52a. The same MHC allele is strongly associated with FNAIT. As the production of pathological antibodies reactive with fetal platelets is likely dependent on these T cell responses, there exists a potential for preventing FNAIT by targeting these T cells.

Preclinical evaluation of immunotherapeutic regimens for fetal/neonatal alloimmune thrombocytopenia.

Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening bleeding disorder caused by maternal antibodies directed against paternally inherited antigens present on the surface of fetal platelets. The human platelet alloantigen HPA-1a (formerly known as the PlA1 alloantigen), is the most frequently implicated HPA for causing FNAIT in Whites. A single Leu33Pro amino acid polymorphism residing within the ∼50-amino-acid plexin-semaphorin-integrin domain near the N-terminus of the integrin ß3 subunit (platelet membrane glycoprotein IIIa [GPIIIa]) is responsible for generating the HPA-1a and HPA-1b epitopes in human GPIIIa and serves as the central target for alloantibody-mediated platelet destruction. To simulate the etiology of human FNAIT, wild-type female mice were pre-immunized with platelets derived from transgenic mice engineered to express the human HPA-1a epitope on a murine GPIIIa backbone. These mice developed a strong alloimmune response specific for HPA-1a, and when bred with HPA-1a+ males, gave birth to severely thrombocytopenic pups that exhibited an accompanying bleeding phenotype. Administering either polyclonal intravenous immunoglobulin G or a human monoclonal blocking antibody specific for the HPA-1a epitope into pregnant female mice resulted in significant elevation of the neonatal platelet count, normalized hemostasis, and prevented bleeding. The establishment of an alloantigen-specific murine model that recapitulates many of the clinically important features of FNAIT should pave the way for the preclinical development and testing of novel therapeutic and prophylactic modalities to treat or prevent FNAIT in humans.

Quantitative MAIPA: Comparison of different MAIPA protocols.

INTRODUCTION: In a screening setting, maternal anti-HPA 1a antibody level has been found to be a good prognostic tool to identify newborns at risk for severe NAIT. AIM: Identify the optimal MAIPA protocol for quantitation of anti-HPA 1a antibodies. MATERIALS AND METHODS: Plasma were analysed for anti-HPA 1a antibodies using different monoclonal antibodies, lyophilized or fresh platelets and MAIPA protocols. RESULTS: The anti-HPA 1a antibody level varied significantly when different monoclonal antibodies were used. However, there was a strong correlation between maternal anti-HPA 1a antibody level and platelet count in the newborn. The sensitivity of the assay depended on the adopted MAIPA protocol. CONCLUSION: Consistent tests results are of importance for the clinical impact of the test.

Is it time to implement HPA-1 screening in pregnancy?

PURPOSE OF REVIEW: The purpose of the review is to argue for and against introduction of HPA-1 typing of all pregnant women to reduce morbidity and mortality caused by foetal/neonatal alloimmune thrombocytopenia (FNAIT). RECENT FINDING: Several groups have done HPA-1 typing in cohorts of pregnant women. Results from a Norwegian study (>100,000 pregnancies) indicate that screening combined with simple intervention decreases morbidity and mortality due to FNAIT and is cost effective in Norway. Results from this study and several other studies show that there is correlation between the level of anti-HPA-1a antibodies in the mother and the severity of thrombocytopenia in the newborn. An important finding is that about 75% of women with antibodies are immunized in connection with delivery. Only 25% of the women are immunized during pregnancy. SUMMARY: Screening for FNAIT does not fully meet the criteria presented by the WHO. Nevertheless, the results of the Norwegian study strongly indicate that morbidity and mortality related to FNAIT can be reduced. If the recent attempts to make a vaccine aimed at prevention of immunization and/or tolerizing peptides or neutralizing antibodies for already immunized women are shown to be successful, screening must be implemented.