Ab Epitope Research Articles

Analyzing the Human Serum Antibody Responses to a Live Attenuated Tetravalent Dengue Vaccine Candidate.

Dengue virus serotypes 1-4 (DENV-1-4) are the most common vector-borne viral pathogens of humans and the etiological agents of dengue fever and dengue hemorrhagic syndrome. A live-attenuated tetravalent dengue vaccine (TDV) developed by Takeda Vaccines has recently progressed to phase 3 safety and efficacy evaluation. We analyzed the qualitative features of the neutralizing antibody (nAb) response induced in naive and DENV-immune individuals after TDV administration. Using DENV-specific human monoclonal antibodies (mAbs) and recombinant DENV displaying different serotype-specific Ab epitopes, we mapped the specificity of TDV-induced nAbs against DENV-1-3. Nearly all subjects had high levels of DENV-2-specific nAbs directed to epitopes centered on domain III of the envelope protein. In some individuals, the vaccine induced nAbs that tracked with a DENV-1-specific neutralizing epitope centered on domain I of the envelope protein. The vaccine induced binding Abs directed to a DENV-3 type-specific neutralizing epitope, but findings of mapping of DENV-3 type-specific nAbs were inconclusive. Here we provide qualitative measures of the magnitude and epitope specificity of the nAb responses to TDV. This information will be useful for understanding the performance of TDV in clinical trials and for identifying correlates of protective immunity.

Molecular characterization of the β-amyloid(4-10) epitope of plaque specific Aβ antibodies by affinity-mass spectrometry using alanine site mutation.

Alzheimer disease is a neurodegenerative disease affecting an increasing number of patients worldwide. Current therapeutic strategies are directed to molecules capable to block the aggregation of the β-amyloid(1-42) (Aβ) peptide and its shorter naturally occurring peptide fragments into toxic oligomers and amyloid fibrils. Aβ-specific antibodies have been recently developed as powerful antiaggregation tools. The identification and functional characterization of the epitope structures of Aβ antibodies contributes to the elucidation of their mechanism of action in the human organism. In previous studies, the Aβ(4-10) peptide has been identified as an epitope for the polyclonal anti-Aβ(1-42) antibody that has been shown capable to reduce amyloid deposition in a transgenic Alzheimer disease mouse model. To determine the functional significance of the amino acid residues involved in binding to the antibody, we report here the effects of alanine single-site mutations within the Aβ-epitope sequence on the antigen-antibody interaction. Specific identification of the essential affinity preserving mutant peptides was obtained by exposing a Sepharose-immobilized antibody column to an equimolar mixture of mutant peptides, followed by analysis of bound peptides using high-resolution MALDI-Fourier transform-Ion Cyclotron Resonance mass spectrometry. For the polyclonal antibody, affinity was preserved in the H6A, D7A, S8A, and G9A mutants but was lost in the F4, R5, and Y10 mutants, indicating these residues as essential amino acids for binding. Enzyme-linked immunosorbent assays confirmed the binding differences of the mutant peptides to the polyclonal antibody. In contrast, the mass spectrometric analysis of the mutant Aβ(4-10) peptides upon affinity binding to a monoclonal anti-Aβ(1-17) antibody showed complete loss of binding by Ala-site mutation of any residue of the Aβ(4-10) epitope. Surface plasmon resonance affinity determination of wild-type Aβ(1-17) to the monoclonal Aβ antibody provided a binding constant KD in the low nanomolar range. These results provide valuable information in the elucidation of the binding mechanism and the development of Aβ-specific antibodies with improved therapeutic efficacy.

Amino-Terminal β-Amyloid Antibody Blocks β-Amyloid-Mediated Inhibition of the High-Affinity Choline Transporter CHT.

Alzheimer’s disease (AD) is a common age-related neurodegenerative disorder that is characterized by progressive cognitive decline. The deficits in cognition and attentional processing that are observed clinically in AD are linked to impaired function of cholinergic neurons that release the neurotransmitter acetylcholine (ACh). The high-affinity choline transporter (CHT) is present at the presynaptic cholinergic nerve terminal and is responsible for the reuptake of choline produced by hydrolysis of ACh following its release. Disruption of CHT function leads to decreased choline uptake and ACh synthesis, leading to impaired cholinergic neurotransmission. We report here that cell-derived β-amyloid peptides (Aβ) decrease choline uptake activity and cell surface CHT protein levels in SH-SY5Y neural cells. Moreover, we make the novel observation that the amount of CHT protein localizing to early endosomes and lysosomes is decreased significantly in cells that have been treated with cell culture medium that contains Aβ peptides released from neural cells. The Aβ-mediated loss of CHT proteins from lysosomes is prevented by blocking lysosomal degradation of CHT with the lysosome inhibitor bafilomycin A1 (BafA1). BafA1 also attenuated the Aβ-mediated decrease in CHT cell surface expression. Interestingly, however, lysosome inhibition did not block the effect of Aβ on CHT activity. Importantly, neutralizing Aβ using an anti-Aβ antibody directed at the N-terminal amino acids 1–16 of Aβ, but not by an antibody directed at the mid-region amino acids 22–35 of Aβ, attenuates the effect of Aβ on CHT activity and trafficking. This indicates that a specific N-terminal Aβ epitope, or specific conformation of soluble Aβ, may impair CHT activity. Therefore, Aβ immunotherapy may be a more effective therapeutic strategy for slowing the progression of cognitive decline in AD than therapies designed to promote CHT cell surface levels.

Plant-based chimeric HPV-virus-like particles bearing amyloid-β epitopes elicit antibodies able to recognize amyloid plaques in APP-tg mouse and Alzheimer's disease brains.

The main amyloid-beta (Aβ) variants detected in the human brain are full-length Aβ1-40 and Aβ1-42 peptides; however, a significant proportion of AD brain Aβ consists also of N-terminal truncated/modified species. The majority of the previous immunotherapeutic strategies targeted the N-terminal immunodominant epitope of the full-length Aβ; however, most of the pathological N-truncated forms of Aβ lack this critical B cell epitope. Recently, virus-like particles (VLPs), self-assembled structures with highly ordered repetitive patterns on their surface and capable of inducing robust immune responses, were applied as a promising platform for various antigen expressions. In this study, we expressed in plants two chimeric HPV16 L1 capsid proteins obtained by introduction of the β-amyloid 11-28 epitope (Aβ 11-28) into the h4 helix or into the coil regions of the L1 protein. The Aβ 11-28 epitope was chosen because it is present in the full-length Aβ 1-42 as well as in the truncated/modified amyloid peptide species. After expression, we assembled the chimerical L1/Aβ 11-28 into a VLP in which the Aβ 11-28 epitope is exposed at very high density (360 times) on the surface of the VLP. The chimeric VLPs elicited in mice Aβ-specific antibodies binding to β-amyloid plaques in APP-tg mouse and AD brains. Our study is the first to demonstrate a successful production in plants and immunogenic properties in mice of chimeric HPV16 L1 VLPs bearing Aβ epitope that may be of potential relevance for the development of multivalent vaccines for a multifactorial disease such as AD.

Antibody Distance from the Cell Membrane Regulates Antibody Effector Mechanisms.

Immunotherapy using mAbs, such as rituximab, is an established means of treating hematological malignancies. Abs can elicit a number of mechanisms to delete target cells, including complement-dependent cytotoxicity, Ab-dependent cellular cytotoxicity, and Ab-dependent cellular phagocytosis. The inherent properties of the target molecule help to define which of these mechanisms are more important for efficacy. However, it is often unclear why mAb binding to different epitopes within the same target elicits different levels of therapeutic activity. To specifically address whether distance from the target cell membrane influences the aforementioned effector mechanisms, a panel of fusion proteins consisting of a CD20 or CD52 epitope attached to various CD137 scaffold molecules was generated. The CD137 scaffold was modified through the removal or addition of cysteine-rich extracellular domains to produce a panel of chimeric molecules that held the target epitope at different distances along the protein. It was shown that complement-dependent cytotoxicity and Ab-dependent cellular cytotoxicity favored a membrane-proximal epitope, whereas Ab-dependent cellular phagocytosis favored an epitope positioned further away. These findings were confirmed using reagents targeting the membrane-proximal or -distal domains of CD137 itself before investigating these properties in vivo, where a clear difference in the splenic clearance of transfected tumor cells was observed. Together, this work demonstrates how altering the position of the Ab epitope is able to change the effector mechanisms engaged and facilitates the selection of mAbs designed to delete target cells through specific effector mechanisms and provide more effective therapeutic agents.

Monoclonal Antibodies against Epidermal Growth Factor Receptor Acquire an Ability To Kill Tumor Cells through Complement Activation by Mutations That Selectively Facilitate the Hexamerization of IgG on Opsonized Cells.

Triggering of the complement cascade induces tumor cell lysis via complement-dependent cytotoxicity (CDC) and attracts and activates cytotoxic cells. It therefore represents an attractive mechanism for mAb in cancer immunotherapy development. The classical complement pathway is initiated by IgG molecules that have assembled into ordered hexamers after binding their Ag on the tumor cell surface. The requirements for CDC are further impacted by factors such as Ab epitope, valency, and affinity. Thus, mAb against well-validated solid tumor targets, such as the epidermal growth factor receptor (EGFR) that effectively induces complement activation and CDC, are highly sought after. The potency of complement activation by IgG Abs can be increased via several strategies. We identified single-point mutations in the Fc domain (e.g., E345K or E430G) enhancing Fc:Fc interactions, hexamer formation, and CDC after Ab binds cell-surface Ag. We show that EGFR Abs directed against clinically relevant epitopes can be converted into mAb with unprecedented CDC activity. Alternative strategies rely on increasing the affinity of monomeric IgG for C1q by introduction of a quadruple mutation at the C1q binding site or via generation of an IgG1/IgG3 chimera. In this study we show that selective enhancement of C1q binding via avidity modulation is superior to the unattended increase in C1q binding via affinity approaches, particularly for target cells with reduced EGFR expression levels. Improving Fc:Fc interactions of Ag-bound IgG therefore represents a highly promising and novel approach for potentiating the anti-tumor activity of therapeutic mAb against EGFR and potentially other tumor targets.

Norovirus P particle-based active A\u03b2 immunotherapy elicits sufficient immunogenicity and improves cognitive capacity in a mouse model of Alzheimer\u2019s disease

Disease-modifying immunotherapies focusing on reducing amyloid-beta (Aβ) deposition are the main treatment for Alzheimer’s disease (AD). However, none of the Aβ immunotherapies has produced clinically meaningful results to date. The main reason for this lack of efficacy is that the vaccine induces insufficiently high antibody titers, as it contains small B-cell epitope of Aβ to avoid Aβ42-specific T-cell activation. With the aim of generating a potent AD vaccine, we designed the protein PP-3copy-Aβ1-6-loop123, comprising three copies of Aβ1-6 inserted into three loops of a novel vaccine platform, the norovirus P particle, which could present Aβ at its surface and remarkably enhance the immunogenicity of the vaccine. We demonstrated that PP-3copy-Aβ1-6-loop123 was able to elicit high antibody titers against Aβ42, without causing T-cell activation, in AD mice regardless of their age. Importantly, PP-3copy-Aβ1-6-loop123 treatment successfully reduced amyloid deposition, rescued memory loss, and repaired hippocampus damage in AD mice. The Aβ antibodies induced by this active immunotherapy reacted with and disrupted aggregated Aβ, reducing its cellular toxicity. In addition, our results suggested PP-3copy-Aβ1-6-loop123 immunization could restore Aβ42 homeostasis in both the serum and brain. Thus, the P particle-based Aβ epitope vaccine is a sufficiently immunogenic and safe immunotherapeutic intervention for Alzheimer’s disease.

Clinical Development of Aducanumab, an Anti-Aβ Human Monoclonal Antibody Being Investigated for the Treatment of Early Alzheimer's Disease.

The amyloid hypothesis has been the dominant framework for Alzheimer's disease (AD) research, including the development of anti-AD therapies. However, none of the phase III clinical trials conducted to date that targeted amyloid β (Aβ) production, aggregation, or clearance demonstrated a statistically significant treatment effect in patients with AD. This includes the approach of using monoclonal antibodies that recognize various Aβ epitopes and display different binding selectivity. While some monoclonal antibodies have failed in phase III trials, several are still in development. Aducanumab (BIIB037) is a human antibody that selectively targets aggregated forms of Aβ, including soluble oligomers and insoluble fibrils. In PRIME (NCT01677572), an ongoing phase Ib trial (N=196 patients dosed), aducanumab was shown to reduce Aβ plaques and slow decline in clinical measures in patients with prodromal or mild AD, with acceptable safety and tolerability. The main safety finding was amyloid-related imaging abnormalities (ARIA), a side effect associated with removal of Aβ, which was dose-dependent and occurred more often in ApoE ε4 carriers than non-carriers. ENGAGE (NCT02477800) and EMERGE (NCT02484547), the ongoing phase III trials of aducanumab in early AD, have been designed based on the outcomes of PRIME and on lessons from past clinical trials in patients with AD. Those study design features include patient selection with confirmed Aβ pathology, ensuring sufficient target engagement, and conducting clinical trials in patients at earlier symptomatic stages of AD.

ELUCIDATION OF NOVEL AB EPITOPE TARGETS ALLOWS FOR AN ANTIBODY-SPECIFIC TARGETING OF EARLY AB OLIGOMERIZATION

ELUCIDATION OF NOVEL AB EPITOPE TARGETS ALLOWS FOR AN ANTIBODY-SPECIFIC TARGETING OF EARLY AB OLIGOMERIZATION

Molecular basis for mid-region amyloid-β capture by leading Alzheimer's disease immunotherapies.

Solanezumab (Eli Lilly) and crenezumab (Genentech) are the leading clinical antibodies targeting Amyloid-β (Aβ) to be tested in multiple Phase III clinical trials for the prevention of Alzheimer's disease in at-risk individuals. Aβ capture by these clinical antibodies is explained here with the first reported mid-region Aβ-anti-Aβ complex crystal structure. Solanezumab accommodates a large Aβ epitope (960 Å2 buried interface over residues 16 to 26) that forms extensive contacts and hydrogen bonds to the antibody, largely via main-chain Aβ atoms and a deeply buried Phe19-Phe20 dipeptide core. The conformation of Aβ captured is an intermediate between observed sheet and helical forms with intramolecular hydrogen bonds stabilising residues 20–26 in a helical conformation. Remarkably, Aβ-binding residues are almost perfectly conserved in crenezumab. The structure explains the observed shared cross reactivity of solanezumab and crenezumab with proteins abundant in plasma that exhibit this Phe-Phe dipeptide.

LBP05: B CELL POSITIVE FLOWCYTOMETRY CROSS MATCH DUE TO DONOR SPECIFIC ANTIBODIES OF IgM ISOTYPE

Aim Analysis of HLA specific antibodies (Ab) is an important component of monitoring of transplant recipients (TR). It includes Ab concentration, epitope specificity, isotype and ability to bind complement. In this study, we have reported positive B lymphocyte flow cytometry cross match (FC CM) owing to donor specific Ab (DSA) of IgM isotype directed to HLA-DQ7. Methods Analysis of anti-HLA Ab was performed in DTT treated serum using single antigen bead (SAB) Luminex technology. Complement binding activity of DSA was investigated using C1q solid phase assay (One Lambda, Inc). PCR-SSOP method was used for HLA typing (One Lambda, Inc). CM of TR serum with donor lymphocytes was performed in the following modifications: T cells CDC-AHG (±DTT), B cells CDC x4 washes (± DTT). B cell CM was performed at 4 °C and room temperatures. FC CM was performed as tri-color assay using secondary FITC-conjugated Ab to human IgG or IgM. In order to block falsely positive reactivity due to surface IgM on the B cells, blocking titration assay using unlabeled anti-human IgM was performed. Results Second kidney TR was typed: A2, 2; B44, 60; DR4, 13; DQ8, 6; DR53, 52. First living donor (LD) was typed A2, 25; B18, 60; DR4, 13; DQ7, 6; DR53,52, and current LD was typed A2,68; B65,60; DR13,13; DQ7,6; DR52,52. At the time of workup broad specificity of anti-Class I Ab was detected, whereas anti-class II reactivity was represented by anti-DQ7 Ab only. The strongest blocking activity of unlabeled anti-human IgM Ab was observed at 40.0 μg/ml concentration. The results of CM assays and Ab analysis are presented in the Table 1 below. a – MFI, mean fluorescence intensity, b – ASHI score, c – MCS, median channel shift, d – tested by Luminex screening IgM assay. Conclusions These findings account for B cell IgG negative CDC CM results despite the high level of DSA. We conclude that under certain circumstances detection of DSA of IgM isotype may provide new insights regarding transplantation in patients presenting with negative B-cell CDC and positive FC CM.

Diversifying Selection Analysis Predicts Antigenic Evolution of 2009 Pandemic H1N1 Influenza A Virus in Humans.

Although a large number of immune epitopes have been identified in the influenza A virus (IAV) hemagglutinin (HA) protein using various experimental systems, it is unclear which are involved in protective immunity to natural infection in humans. We developed a data mining approach analyzing natural H1N1 human isolates to identify HA protein regions that may be targeted by the human immune system and can predict the evolution of IAV. We identified 16 amino acid sites experiencing diversifying selection during the evolution of prepandemic seasonal H1N1 strains and found that 11 sites were located in experimentally determined B-cell/antibody (Ab) epitopes, including three distinct neutralizing Caton epitopes: Sa, Sb, and Ca2 [A. J. Caton, G. G. Brownlee, J. W. Yewdell, and W. Gerhard, Cell 31:417-427, 1982, http://dx.doi.org/10.1016/0092-8674(82)90135-0]. We predicted that these diversified epitope regions would be the targets of mutation as the 2009 H1N1 pandemic (pH1N1) lineage evolves in response to the development of population-level protective immunity in humans. Using a chi-squared goodness-of-fit test, we identified 10 amino acid sites that significantly differed between the pH1N1 isolates and isolates from the recent 2012-2013 and 2013-2014 influenza seasons. Three of these sites were located in the same diversified B-cell/Ab epitope regions as identified in the analysis of prepandemic sequences, including Sa and Sb. As predicted, hemagglutination inhibition (HI) assays using human sera from subjects vaccinated with the initial pH1N1 isolate demonstrated reduced reactivity against 2013-2014 isolates. Taken together, these results suggest that diversifying selection analysis can identify key immune epitopes responsible for protective immunity to influenza virus in humans and thereby predict virus evolution. The WHO estimates that approximately 5 to 10% of adults and 20 to 30% of children in the world are infected by influenza virus each year. While an adaptive immune response helps eliminate the virus following acute infection, the virus rapidly evolves to evade the established protective memory immune response, thus allowing for the regular seasonal cycles of influenza virus infection. The analytical approach described here, which combines an analysis of diversifying selection with an integration of immune epitope data, has allowed us to identify antigenic regions that contribute to protective immunity and are therefore the key targets of immune evasion by the virus. This information can be used to determine when sequence variations in seasonal influenza virus strains have affected regions responsible for protective immunity in order to decide when new vaccine formulations are warranted.

Structural biology of Alzheimer's disease

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease in humans with age being the biggest risk factor. The mechanisms by which the disease progresses to cognitive decline in the sufferer are complex and not fully elucidated. A defining pathological feature is the deposition of extracellular plaques composed primarily of misfolded amyloid beta (Aβ) peptide: a proteolytic breakdown product of the much larger Amyloid Precursor Protein. While Aβ peptides are the main constituents of amyloid plaques that burden the diseased brain, plaque burden correlates poorly with the severity of the disease. There is accumulating evidence that a prefibrillar or protofibrillar soluble form of Aβ can compromise neuronal functions and trigger cell death. Immunotherapy targeting Abeta is a promising direction in AD research with active and passive immunotherapies shown to lower cerebral Aβ levels and rescue cognitive function in animal models. Anti-Aβ immunotherapies are a significant class of AD therapeutics currently in human clinical trials. We have been examining the molecular basis of antibody engagement of Aβ epitopes to inform the analysis of clinical trial data and to guide the engineering of anti-Aβ antibodies with optimised specificity and affinity. We have determined the structures of three different AD antibodies in complex with Ab peptides: (1) WO2, which recognises the N-terminus of Aβ, (2) Mab 2286, which like the AD immunotherapeutic Ponezumab (Pfizer), shows specificity for the C-terminus of Aβ40 but has no significant cross-reactivity with Aβ42/43, and (3) Bapineuzumab, a humanized antibody developed by Pfizer and Johnson & Johnson which recognises the N-terminus of Aβ but cannot recognize N-terminally modified or truncated Aβ peptides (1). All these studies reveal surprising aspects of Aβ peptide recognition by the antibodies and suggest new avenues for AD antibody development.

IgG-mediated anaphylaxis to a synthetic long peptide vaccine containing a B cell epitope can be avoided by slow-release formulation.

Synthetic long peptides (SLP) are a promising vaccine modality to induce therapeutic T cell responses in patients with chronic infections and tumors. We studied different vaccine formulations in mice using SLP derived from carcinoembryonic Ag. We discovered that one of the SLP contains a linear Ab epitope in combination with a CD4 epitope. Repeated vaccination with this carcinoembryonic Ag SLP in mice shows improved T cell responses and simultaneously induced high titers of peptide-specific Abs. These Abs resulted in unexpected anaphylaxis after a third or subsequent vaccinations with the SLP when formulated in saline. Administration of low SLP doses in the slow-release vehicle IFA prevented the anaphylaxis after repeated vaccination. This study underscores both the immunogenicity of SLP vaccination, for inducing T cell as well as B cell responses, and the necessity of safe administration routes.

Epitope-based DNA vaccine for Alzheimer's disease: Translational study in macaques

BackgroundClinical trials with passive and active Alzheimer's disease (AD) vaccines suggest that early interventions are needed for improvement of cognitive and/or functional performance in patients, providing impetus for the development of safe and immunologically potent active vaccines targeting amyloid β (Aβ). The AN-1792 trial has indicated that Aβ-specific T cells may be unsafe for humans; therefore, other vaccines based on small Aβ epitopes are undergoing preclinical and clinical testing. MethodsHumoral and cellular immune responses elicited in response to a novel DNA epitope-based vaccine (AV-1955) delivered to rhesus macaques using the TriGrid electroporation device were evaluated. Functional activities of anti-Aβ antibodies generated in response to vaccination were assessed in vitro. ResultsAV-1955 generates long-term, potent anti-Aβ antibodies and cellular immune responses specific to foreign T-helper epitopes but not to self-Aβ. ConclusionsThis translational study demonstrates that a DNA-based epitope vaccine for AD could be appropriate for human clinical testing.

Systemic Lupus Erythematosus Patients Contain Significantly Less IgM against Mono-Methylated Lysine than Healthy Subjects

Post-translational modifications on proteins are important in biological processes but may create neo-epitopes that induce autoimmune responses. In this study, we measured the serum IgG and IgM response to a set of non-modified or acetyl- and methyl-modified peptides corresponding to residues 1–19 of the histone 3 N-terminal tail in systemic lupus erythematosus (SLE) patients and healthy subjects. Our results indicated that the SLE patients and healthy subjects produced antibodies (Abs) to the peptides, but the two groups had different Ab isotype and epitope preferences. Abs to the non-modified form, H31–19, were of the IgG isotype and produced by SLE patients. They could not recognize the scrambled H31–19, which contained the same amino acid composition but a different sequence as H31–19. In comparison, healthy subjects in general did not produce IgG against H31–19. However, about 70% of the healthy subjects produced IgM Abs against mono-methylated K9 of H31–19 (H31–19K9me). Our further studies revealed that ε-amine mono-methylated lysine could completely inhibit the IgM binding to H31–19K9me, but lysine had no inhibitory effect. In addition, the IgM Abs could bind peptides containing a mono-methylated lysine residue but with totally different sequences. Thus, mono-methylated lysine was the sole epitope for the IgM. Interestingly, SLE patients had much lower levels of this type of IgM. There was no obvious correlation between the IgM levels and disease activity and the decreased IgM was unlikely caused by medical treatments.We also found that the IgM Abs were not polyreactive to dsDNA, ssDNA, lipopolysaccharide (LPS) or insulin and they did not exist in umbilical cord serum, implying that they were not natural Abs. The IgM Abs against mono-methylated lysine are present in healthy subjects but are significantly lower in SLE patients, suggesting a distinct origin of production and special physiological functions.

Novel Biopanning Strategy To Identify Epitopes Associated with Vaccine Protection

Identifying immune correlates of protection is important to develop vaccines against infectious diseases. We designed a novel, universally applicable strategy to profile the antibody (Ab) repertoire of protected vaccine recipients, using recombinant phages encoding random peptide libraries. The new approach, termed "protection-linked (PL) biopanning," probes the Ab paratopes of protected vaccinees versus those with vaccine failure. As proof of concept, we screened plasma samples from vaccinated rhesus macaques (RMs) that had completely resisted multiple mucosal challenges with R5-tropic simian-human immunodeficiency viruses (SHIVs). The animals had been immunized with a multicomponent vaccine (multimeric HIV-1 gp160, HIV-1 Tat, and SIV Gag-Pol particles). After PL biopanning, we analyzed the phagotopes selected for amino acid homologies; in addition to the expected Env mimotopes, one recurring motif reflected the neutralizing Ab epitope at the N terminus (NT) of HIV-1 Tat. Subsequent binding and functional assays indicated that anti-Tat NT Abs were present only in completely or partially protected RMs; peak viremia of the latter was inversely correlated with anti-Tat NT Ab titers. In contrast, highly viremic, unvaccinated controls did not develop detectable Abs against the same epitope. Based upon the protective effect observed in vivo, we suggest that Tat should be included in multicomponent HIV-1 vaccines. Our data highlight the power of the new PL-biopanning strategy to identify Ab responses with significant association to vaccine protection, regardless of the mechanism(s) or targets of the protective Abs. PL biopanning is also unbiased with regard to pathogens or disease model, making it a universal tool.

Epitope structure and binding affinity of single chain llama anti‐β‐amyloid antibodies revealed by proteolytic excision affinity‐mass spectrometry

ß-Amyloid (Aß) immunotherapy has become a promising strategy for reducing the level of Aß in brain. New immunological approaches have been recently proposed for rapid, early diagnosis, and molecular treatment of neurodegenerative diseases related to Alzheimer's Disease (AD). The combination of proteolytic epitope excision and extraction and mass spectrometry using digestion with various proteases has been shown to be an efficient tool for the identification and molecular characterization of antigenic determinants. Here, we report the identification of the Aβ epitope recognized by the variable domain of single chain llama anti-Aβ-antibodies, termed Aβ-nanobodies, that have been discovered in the blood of camelids and found to be promising candidates for immunotherapy of AD. The epitope recognized by two Aβ-specific nanobodies was identified by proteolytic epitope extraction- and excision-mass spectrometry using a series of proteases (trypsin, chymotrypsin, GluC-protease, and LysC-protease). Matrix-assisted laser desorption ionization--mass spectrometric analysis of the affinity--elution fraction provided the epitope, Aβ(17-28), in the mid- to carboxy-terminal domain of Aβ, which has been shown to exert an Aß-fibril inhibiting effect. Affinity studies of the synthetic epitope confirmed that the Aβ(17-28) peptide is the minimal fragment that binds to the nanobodies. The interactions between the nanobodies and full length Aβ(1-40) or Aβ-peptides containing or lacking the epitope sequence were further characterized by enzyme linked immunosorbent assay and bioaffinity analysis. Determinations of binding affinities between the Aβ-nanobodies and Aβ(1-40) and the Aβ(17-28) epitope provided K(D) values of approximately 150 and 700 nmol, respectively. Thus, the knowledge of the epitope may be highly useful for future studies of Aβ-aggregation (oligomerization and fibril formation) and for designing new aggregation inhibitors.

Active DNA Aβ42 vaccination as immunotherapy for Alzheimer disease

As a neurodegenerative disorder, Alzheimer disease (AD) is the most common form of dementia found in the aging population. Immunotherapy with passive or active immunizations targeting amyloid beta (Aβ) build-up in the brain may provide a possible treatment option and may help prevent AD from progressing. A number of passive immunizations with anti-Aβ42 antibodies are in different phases of clinical trials. One active immunization approach, AN-1792, was stopped after the development of autoimmune encephalitis in 6% of patients and a second one, CAD106, in which a small Aβ epitope is used, is currently in safety and tolerability studies. Besides active immunizations with proteins or peptides, active immunizations using DNA which codes for the protein against which the immune response will be directed, so called genetic immunizations, provide additional safety as the immune response in DNA immunizations differs quantitatively and qualitatively from the response elicited by peptide immunizations. In this review, we summarize our data using DNA Aβ42 immunizations in mouse models and discuss the results together with the results presented by other groups working on a DNA vaccine as treatment option for AD.

P1‐228: Naturally occurring human antibodies to misfolded Aβ epitope associated with Alzheimer's disease

P1‐228: Naturally occurring human antibodies to misfolded Aβ epitope associated with Alzheimer's disease