Littérature scientifique sur le sujet « Broadly reactive antibody »

ABSTRACT Norwalk virus, a member of the family Caliciviridae, is an important cause of acute epidemic nonbacterial gastroenteritis. Norwalk and related viruses are classified in a separate genus of Caliciviridae called Norovirus, which is comprised of at least three genogroups based on sequence differences. Many of the currently available immunologic reagents used to study these viruses are type specific, which limits the identification of antigenically distinct viruses in detection assays. Identification of type-specific and cross-reactive epitopes is essential for designing broadly cross-reactive diagnostic assays and dissecting the immune response to calicivirus infection. To address this, we have mapped the epitopes on the norovirus capsid protein for both a genogroup I-cross-reactive monoclonal antibody and a genogroup II-cross-reactive monoclonal antibody by use of norovirus deletion and point mutants. The epitopes for both monoclonal antibodies mapped to the C-terminal P1 subdomain of the capsid protein. Although the genogroup I-cross-reactive monoclonal antibody was previously believed to recognize a linear epitope, our results indicate that a conformational component of the epitope explains the monoclonal antibody's genogroup specificity. Identification of the epitopes for these monoclonal antibodies is of significance, as they are components in a commercially available norovirus-diagnostic enzyme-linked immunosorbent assay.

Influenza virus strain-specific monoclonal antibodies (mAbs) provide protection independent of Fc gamma receptor (FcγR) engagement. In contrast, optimal in vivo protection achieved by broadly reactive mAbs requires Fc–FcγR engagement. Most strain-specific mAbs target the head domain of the viral hemagglutinin (HA), whereas broadly reactive mAbs typically recognize epitopes within the HA stalk. This observation has led to questions regarding the mechanism regulating the activation of Fc-dependent effector functions by broadly reactive antibodies. To dissect the molecular mechanism responsible for this dichotomy, we inserted the FLAG epitope into discrete locations on HAs. By characterizing the interactions of several FLAG-tagged HAs with a FLAG-specific antibody, we show that in addition to Fc–FcγR engagement mediated by the FLAG-specific antibody, a second intermolecular bridge between the receptor-binding region of the HA and sialic acid on effector cells is required for optimal activation. Inhibition of this second molecular bridge, through the use of an F(ab′)2or the mutation of the sialic acid-binding site, renders the Fc–FcγR interaction unable to optimally activate effector cells. Our findings indicate that broadly reactive mAbs require two molecular contacts to possibly stabilize the immunologic synapse and potently induce antibody-dependent cell-mediated antiviral responses: (i) the interaction between the Fc of a mAb bound to HA with the FcγR of the effector cell and (ii) the interaction between the HA and its sialic acid receptor on the effector cell. This concept might be broadly applicable for protective antibody responses to viral pathogens that have suitable receptors on effector cells.

ABSTRACT The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) has several adaptations that allow the virus to evade antibody neutralization. Nevertheless, a few broadly cross-reactive neutralizing antibodies as well as reagents containing portions of CD4, the HIV receptor, have demonstrated partial efficacy in suppressing viral replication. One type of reagent designed for improved HIV neutralization fuses the CD4 D1-D2 domains to the variable regions of an antibody recognizing the CD4-induced (CD4i) coreceptor binding site on the gp120 portion of the HIV envelope spike. We designed, expressed, purified, and tested the neutralization potencies of CD4-CD4i antibody reagents with different architectures, antibody combining sites, and linkers. We found that fusing CD4 to the heavy chain of the CD4i antibody E51 yields a bivalent reagent including an antibody Fc region that expresses well, is expected to have a long serum half-life, and has comparable or greater neutralization activity than well-known broadly neutralizing anti-HIV antibodies. A CD4 fusion with the anti-HIV carbohydrate antibody 2G12 also results in a potent neutralizing reagent with more broadly neutralizing activity than 2G12 alone.

Abstract To generate a hepatitis E virus (HEV) genotype 3 (HEV-3)–specific monoclonal antibody (mAb), the Escherichia coli–expressed carboxy-terminal part of its capsid protein was used to immunise BALB/c mice. The immunisation resulted in the induction of HEV-specific antibodies of high titre. The mAb G117-AA4 of IgG1 isotype was obtained showing a strong reactivity with the homologous E. coli, but also yeast-expressed capsid protein of HEV-3. The mAb strongly cross-reacted with ratHEV capsid protein derivatives produced in both expression systems and weaker with an E. coli–expressed batHEV capsid protein fragment. In addition, the mAb reacted with capsid protein derivatives of genotypes HEV-2 and HEV-4 and common vole hepatitis E virus (cvHEV), produced by the cell-free synthesis in Chinese hamster ovary (CHO) and Spodoptera frugiperda (Sf21) cell lysates. Western blot and line blot reactivity of the mAb with capsid protein derivatives of HEV-1 to HEV-4, cvHEV, ratHEV and batHEV suggested a linear epitope. Use of truncated derivatives of ratHEV capsid protein in ELISA, Western blot, and a Pepscan analysis allowed to map the epitope within a partially surface-exposed region with the amino acid sequence LYTSV. The mAb was also shown to bind to human patient–derived HEV-3 from infected cell culture and to hare HEV-3 and camel HEV-7 capsid proteins from transfected cells by immunofluorescence assay. The novel mAb may serve as a useful tool for further investigations on the pathogenesis of HEV infections and might be used for diagnostic purposes. Key points • The antibody showed cross-reactivity with capsid proteins of different hepeviruses. • The linear epitope of the antibody was mapped in a partially surface-exposed region. • The antibody detected native HEV-3 antigen in infected mammalian cells.

ABSTRACT In addition to the direct neutralization of virus, there is a broader potential for antibody-mediated inhibition of human immunodeficiency virus (HIV) by targeting HIV to effector cells. We demonstrate here that a bispecific antibody incorporating a broadly reactive anti-gp41 antibody, F240, and an anti-IgA receptor (CD89) antibody is effective at directing neutrophils to destroy HIV. Not only are neutrophils the predominant type of white blood cells and very efficient at mediating cell cytotoxicity, they are relatively resistant to infection with HIV. Therefore, they represent a significant weapon against infection if they can be directed and armed to destroy HIV and infected cells.

ABSTRACTEmerging H7N9 influenza virus infections in Asia have once more spurred the development of effective prepandemic H7 vaccines. However, many vaccines based on avian influenza viruses—including H7—are poorly immunogenic, as measured by traditional correlates of protection. Here we reevaluated sera from an H7N1 human vaccine trial performed in 2006. We examined cross-reactive antibody responses to divergent H7 strains, including H7N9, dissected the antibody response into head- and stalk-reactive antibodies, and tested thein vivopotency of these human sera in a passive-transfer H7N9 challenge experiment with mice. Although only a low percentage of vaccinees induced neutralizing antibody responses against the homologous vaccine strain and also H7N9, we detected strong cross-reactivity to divergent H7 hemagglutinins (HAs) in a large proportion of the cohort with a quantitative enzyme-linked immunosorbent assay. Furthermore, H7N1 vaccination induced antibodies to both the head and stalk domains of the HA, which is in sharp contrast to seasonal inactivated vaccines. Finally, we were able to show that both neutralizing and nonneutralizing antibodies improvedin vivovirus clearance in a passive-transfer H7N9 challenge mouse model.

ABSTRACT IMPACT: This study aims to provide insight into naturally acquired immunity against severe malaria, thereby laying the foundation for the design of novel vaccine candidates to prevent severe disease as well as monoclonal antibody therapies to treat severe malaria. OBJECTIVES/GOALS: Severe malaria is caused by parasite surface antigens that contain high sequence diversity. Nevertheless, P. falciparum-exposed individuals develop antibody responses against these antigens. Our goal is to isolate antibodies with broad reactivity to understand how disease protection is acquired. METHODS/STUDY POPULATION: Our study cohort consists of Ugandan adults living in a malaria-endemic region with high transmission intensity, who are protected against severe malaria. Using fluorescently labeled probes of parasite surface antigens, we have isolated antigen-specific B cells from these donors. We then expressed the corresponding monoclonal antibodies in vitro. These antibodies were screened against a library of variant surface antigens to determine antibody breadth and potential to inhibit interaction of the parasite surface antigen with host receptors, a critical step in pathogenesis. Additionally, using a panel of variant surface antigen mutants, we have predicted the epitopes targeted by the broadest monoclonal antibodies. RESULTS/ANTICIPATED RESULTS: We have identified three monoclonal antibodies with exceptionally broad reactivity and inhibitory activity against our panel of severe disease-inducing variant surface antigens. We have identified two major sites targeted by these broadly reactive antibodies. The first site was associated with the largest breadth, but limited inhibitory potential, while the second site showed high-affinity antibody binding and inhibition of receptor binding. Interestingly, two of these three antibodies were very similar in structure, even though they were isolated from different donors. Isolation of antigen-specific B cells from additional donors will enable us to identify how common such broadly reactive antibodies are and allow the identification of additional epitopes DISCUSSION/SIGNIFICANCE OF FINDINGS: This study is the first to isolate broadly reactive antibodies that are likely to protect against severe malaria in naturally immune individuals. Further characterization of antibody-antigen interactions will inform the development of this surface antigen as a vaccine candidate for malaria.

ABSTRACTHighly pathogenic H5N1 virus infection causes severe disease and a high rate of fatality in humans. Development of humanized monoclonal antibodies may provide an efficient therapeutic regime for H5N1 virus infection. In the present study, broadly cross-reactive monoclonal antibodies (MAbs) derived from mice were humanized to minimize immunogenicity. One chimeric antibody (cAb) and seven humanized antibodies (hAbs) were constructed. These antibodies retained broad-spectrum reactivity to H5N1 viruses, binding to recombinant H5-subtype HA1 molecules expressed in CHO cells in a dose-dependent manner and exhibiting similar reactivities against antigenically distinct H5N1 viruses in hemagglutination inhibition (HI) assays. One humanized antibody, 37 hAb, showed HI and neutralization activities comparable to that of the parental murine antibody, 13D4 MAb, while the other six antibodies were less reactive to H5N1 viruses. Analysis of amino acid sequences in the variable region frameworks of the seven humanized antibodies found that Q5 and Y27 in the VH region are highly conserved murine residues. Comparison of the three-dimensional structures derived from the variable regions of MAbs 37 hAb, H1202-34, and 13D4 revealed that residue substitutions at sites 70 and 46 may be the major cause for the observed differences in binding affinity. Examination of the chimeric antibody and one of the humanized antibodies, 37 hAb, showed that both antibodies offered postinfection protection against lethal challenge with antigenically diverse H5N1 viruses in the mouse model. Chimeric and humanized antibodies which retain the broadly reactive and protective properties of murine H5-specific monoclonal antibodies have great potential for use in the treatment of human H5N1 infection.

Influenza Viren stellen eine große Bedrohung der öffentlichen Gesundheit dar. Die saisonale Grippeschutzimpfung induziert Antikörper gegen den Kopfbereich des viralen Oberflächenproteins Hämagglutinin (HA), in dem verstärkt Antigendrift auftritt. Dadurch wird die Effektivität der saisonalen Grippeimpfung auf den Impfstamm beschränkt und es besteht kein ausreichender Schutz gegen virale Driftvarianten. Eine universellere Grippeimpfung wird dringend benötigt. Die Entdeckung breit reaktiver Antikörper gegen den konservierten HA-Stammbereich hat die Erforschung neuartiger Impfstrategien vorangetrieben. Mit Chimären oder Headless HA kann eine Fokussierung der Immunantwort auf immunsubdominante Bereiche im HA-Stammbereich erzielt werden. Auch innovative Impfstoffplattformen wie Adeno-assoziierte Virus (AAV)-Vektoren bergen ein immenses Potenzial, da sie zum einen für die Verwendung im Menschen zugelassen sind und zum anderen die Immunogenität des Antigens positiv beeinflusst. Die Immunisierung mit AAV-Vektoren, die wildtypisches HA, Chimäre HA oder Nukleoprotein exprimieren, führte in dieser Arbeit in Mäusen zur Induktion breit reaktiver Antikörper, nicht aber die Immunisierung mit AAV-Headless HA oder inaktiviertem Grippeimpfstoff. Die AAV-Vektor Impfstoffe führten zur robusten Induktion Fc-Gamma-Rezeptor-aktivierender Antikörper, die beispielsweise Antikörper-vermittelte zelluläre Zytotoxizität auslösen können. Nicht nur die Impfung mit AAV-Chimären HA, sondern auch mit AAV-wildtypischem HA induzierte Antikörper gegen den HA-Stammbereich. Somit kann anscheinend allein durch eine AAV-Vektor vermittelte Expression des Antigens die Immundominanz des HA-Kopfbereiches abgemildert werden. Abschließend konnte zum ersten Mal die Schutzwirkung einer AAV-Vektor Immunisierung gegen HA im Frettchen demonstriert werden. Die in dieser Arbeit beschriebenen Ergebnisse zeigen somit das große Potenzial von AAV-Vektoren als Impfvehikel für eine breit reaktive Grippeschutzimpfung auf.
Influenza viruses represent a severe threat to public health. A seasonal vaccine is available, which readily leads to the induction of antibodies against the head domain of the viral surface protein hemagglutinin (HA), which is prone to antigenic drift. Thus, seasonal vaccination induces only strain specific protection, while it is not effective against drifted virus strains. Hence, there is an urgent need for a universal influenza vaccine. The discovery of broadly reactive antibodies against the highly conserved HA-stalk domain has prompted great interest into research on vaccination strategies to induce broadly protective HA antibodies. Chimeric and headless HA have shown promising results with respect to re-focusing immunity towards immunosubdominant epitopes in the HA-stalk to induce protective HA-stalk antibodies. Also, innovative vaccine delivery platforms such as Adeno-associated virus (AAV)-vectors offer an attractive developmental perspective. AAV-vectors are licensed for use in humans and the AAV-vectored antigen expression positively influences its immunogenicity. In this thesis, immunization with AAV-vectors expressing wildtype HA, chimeric HA or nucleoprotein induced broad protection in mice, but not vaccination with AAV-vectors expressing headless HA or an inactivated influenza vaccine. Protection was associated with the ability of the AAV-vectored vaccines to induce Fc-gamma-receptor-activating antibodies, which might activate antibody-dependent cellular cytotoxicity. Not only chimeric HA but also wildtype HA induced antibodies against the HA-stalk, suggesting that AAV-vectored antigen expression can mitigate the immunodominance of virus strain-specific epitopes in the HA-head. Importantly, for the first time a protective effect AAV-vectored immunization towards HA could be shown in ferrets. Thus, results described in this thesis suggest a large potential for the development of AAV-vectors as carriers for a broadly protective influenza vaccine.

HIV-seropositive homosexuals, narcotic addicts and hemophiliacs develop a new syndrome of immunologic thrombocytopenic purpura (ITP) which is clinically indistinguishable from classic autoimmune thrombocytopenic purpura (ATP) with respect to increased megakaryocytes in the bone marrow, peripheral destruction of antibody-coated platelets, negative serology for SLE, response to treatment with prednisone and/or splenectomy. However, their platelet immunologic profiles are different.Homosexuals appear to have an immune complex-mediated mechanism: markedly elevated platelet-bound IgG and C3C4 (3.8 and 4.2-fold greater than classic ATP, respectively), elevated circulating immune complexes (3-fold greater than classic ATP), anti-F(ab')2 antibodies and absence of 7S anti-platelet IgG. There is no inverse correlation between platelet count and platelet-bound IgG or platelet-elutable anti-platelet antibody as in classic ATP.Hemophiliacs appear to have an autoimmune 7S IgG-mediated mechanism similar to classic ATP: inverse relationship betweem platelet count and platelet-bound IgG, r = 0.84, p less than 0.001, 26 df, anti-platelet reactive 7S IgG which reacts by its F(ab')2 domain, (reactive at 60-130 ug/ml compared to control IgG), platelet-elutable anti-platelet antibody. However, these patients also have elevated circulating immune complexes (2.4-fold classic ATP level) and markedly elevated platelet-bound IgG and C3C4 (3.4 and 1.2-fold classic ATP level, respectively). Anti-HIV antibody correlated with circulating immune complexes, r = 0.833, p less than 0.001.Narcotic addicts appear to have a mixture of both mechanisms (immune complex as well as autoimmune 7S IgG): markedly elevated platelet-bound IgG and C3C4 (2.6 and 2.4-fold classic ATP level, respectively), elevated circulating immune complexes (7.3-fold classic ATP level), anti-F(ab')2 antibodies, absence of an inverse correlation between platelet count and platelet-bound IgG. However, these patients do have specific 7S IgG anti-platelet antibody, which reacts by its F(ab')2 domain.F(ab')2antibodies were of the IgG class and correlated with circulating immune complex level. They react with autologous, homologous patient and healthy control F(ab')2 fragments. Some anti-F(ab')2 antibodies have broad reactivity, others are more limited. Some immune complexes were shown to contain HIV antibody. It is postulated that the immune complex platelet deposition noted with homosexual and narcotic addict thrombocytopenia may in part be due to HIV antibody complexes, some of which may exist as anti-antibody complexes.