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Strategies to guide the affinity maturation process
Zeynep Tellioğlu & Manuel Kenzler Milano, 13/11/2018
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Strategies to guide the affinity maturation process
What we know Antibodies with protective activity are crucial for vaccine efficiency Affinity maturation increases antibody activity through: Somatic Hypermutation (SHM) Selection in the germinal center Affinity maturation: The process that antibodies gain increased affinity and anti-pathogen activity. It is a result of SHM of immunoglobulin genes in B cells coupled to selection for antigen binding 11/13/2018 Unimi - MBC - Molecular Immunology
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Strategies to guide the affinity maturation process
What we know Somatic Hypermutation: Cellular mechanism by which the immune system adapts to new foreign elements. Major component is affinity maturation Germinal Center: Structure within secondary lymphoid tissues Resulting cells are highly mutated from the germline encoded parts and the level of antigen-antibody affinity is increased compared to naïve B cell receptors 11/13/2018 Unimi - MBC - Molecular Immunology
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Unimi - MBC - Molecular Immunology
Overview of B cell activation and SHM MacLeod et al. (2016) B cells are activated by infection and stimulated to divide. During proliferation, SHM occurs. Also, HIV-1 is a highly variable pathogen. 11/13/2018 Unimi - MBC - Molecular Immunology
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Unimi - MBC - Molecular Immunology
Why to guide affinity maturation? For highly variable pathogens (e.g. HIV-1, influenza) vaccination or infection is poorly functional and insufficiently cross-reactive (neutralizing) B cell selection is driven by: affinity to the antigen that is present in the germinal center NOT by functionality that may be desirable in a vaccine Antibodies can perform many antiviral functions including neutralizing of free virus and Fc-receptor requiring mechanisms such as antibody- dependent cell-mediated cytotoxicity (ADCC) Not all the mutations are needed for full activity By vaccination, over 20% is difficult to reach Thus, 5-20% of mutation level is a good percentage to achieve for specific antibodies bB 11/13/2018 Unimi - MBC - Molecular Immunology
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Unimi - MBC - Molecular Immunology
What ‘s Antibody- dependent cell-mediated cytotoxicity ADCC is a mechanism off cell mediated immune defense whereby an effector cell of the immune system (e.g. NK cells) actively lyses a target cell whose membrane surface antigens have been bound by specific antibodies. ADCC is a part of adaptive immune response. bB 11/13/2018 Unimi - MBC - Molecular Immunology
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Unimi - MBC - Molecular Immunology
How to proceed in affinity maturation Naive or memory B cells are activated by vaccination or infection B cells migrate to germinal center via secondary lymphoid tissues such as lymph nodes and they cycle between dark zone and light zone In dark zone, they proliferate and undergo SHM bB 11/13/2018 Unimi - MBC - Molecular Immunology
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Unimi - MBC - Molecular Immunology
How to proceed in affinity maturation In light zone, according to their antigen affinity level, they undergo selection and 90% of the selected B cells return to the dark zone and repeat the cycle. 10% of the B cells exit the cycle to serve as memory B cells or plasma cells 30% of mutation level is reached by infection. Up to 20% mutation level is reached by vaccination and it is sufficient for maturation bB 11/13/2018 Unimi - MBC - Molecular Immunology
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Unimi - MBC - Molecular Immunology
What is the right direction for guiding maturation? Immunogens that can bind to naïve B cell receptors (BCRs) with favorable genetic properties can trigger the initial development of broadly neutralizing antibodies Additionally, there are other sites targeted by broadly neutralizing antibodies in multiple donors that don’t share genetic properties Immunogens that bind to naïve BCRs of multiple unrelated lineages may be more difficult to engineer but would have great potential as vaccines After initial activation of the B cells, further immunization will be required to drive maturation process bB 11/13/2018 Unimi - MBC - Molecular Immunology
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Unimi - MBC - Molecular Immunology
Antibody-virus co-evolution from the origin of the linage through maturation In early infection, autologous neutralizing antibodies develop and neutralize autologous viruses. After the viral escape, they undergo mutations that create epitopes to broadly neutralize both viral strains bB 11/13/2018 Unimi - MBC - Molecular Immunology
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Unimi - MBC - Molecular Immunology
Antibody-virus co-evolution from the origin of the linage through maturation Viral diversification leads to B cell selection that can target a known site of vulnerability and gives autologous neutralization. Following selection of this B cell and virus evolution leads to maturation that results broadly neutralizing antibodies In both approaches, viral sequence diversification was followed by the development of neutralization breadth bB 11/13/2018 Unimi - MBC - Molecular Immunology
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Strategies to guide the affinity maturation process
Immunogen choices Viral Immunogen undergo significant structural rearrangements form can be crucial structural stabilization can improve immunogenicity Immunogens need to present vulnerability sites min. presence of non-neutr. Epitopes HIV-1 Env. Gp120 Many neutralizing epitopes Many non-neutralizing epitopes Tested vaccines elicitate Ab only neutralizing laboratory-adapted strains nNAb compete w bNAb (McGuire et al.) metastable stable McLellan et al. showed Stabilizing prefusion form (via mutation) That locked the RSV in prefusion form 50 times higher levels of neutralizing AB compared to postfusion form. 11/13/2018 Unimi - MBC - Molecular Immunology
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Strategies to guide the affinity maturation process
Immunogen choices HIV-1 immunogens monomeric or trimers (w non HIV-domains) present suboptimal epitopes & low neutralization levels BG505 SOSIP.664 Env trimer (Moore et al.) mimics the prefusion close form binds to bNAb (incl. quaternary epitopes) Structural characterization allow design of further immunogens Improvement of immunogenic response BG505 SOSIP.664 (prefusion form) 11/13/2018 Unimi - MBC - Molecular Immunology
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Strategies to guide the affinity maturation process
Immunogen choices Influenza HA (hemagglutinin) stable trimers available HA head region is antigenically dominant HA head region varies between immunogens limited neutralization Design of HA stem region immunogen cross reactivity Ab (clinical studies) HA head region is antigenically dominant. 11/13/2018 Unimi - MBC - Molecular Immunology
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Strategies to guide the affinity maturation process
Proposed immunization strategies I. Based on Ab ontogeny Prime w modified designed viral proteins engagement of reverted germline versions of known mature Ab Boost w mutants introducing glycans/ sequence variations not recognized by Ab but neutralized by intermediate/ mature Ab Vaccine concepts for induction of cross-reactive Ab II. Glycans not recognized by germline Ab but are neutralized by intermediate/mature Ab 11/13/2018 Unimi - MBC - Molecular Immunology
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Strategies to guide the affinity maturation process
Proposed immunization strategies HIV-1 Highly glycosylated Removing proximal glycans (tailoring the initial immunogen) Open up the area of interest Broad response to area of interest Immunization w more closed immunogens Forcing mutation in Ab Still targeting the area of interest Vaccine concepts for induction of cross-reactive Ab II. Glycans not recognized by germline Ab but are neutralized by intermediate/mature Ab 11/13/2018 Unimi - MBC - Molecular Immunology
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Strategies to guide the affinity maturation process
Proposed immunization strategies II. Based on Ab-viral co-evolution Boost w variants mimic Ab-virus co-evolution pathway recapitulate viral evolution in a single donor Designed immunogens based on early escape variants plus multiple later variants escaped from immune response variants bind to intermediates and mature Ab single variants or combination of variants Vaccine concepts for induction of cross-reactive Ab II. Glycans not recognized by germline Ab but are neutralized by intermediate/mature Ab 11/13/2018 Unimi - MBC - Molecular Immunology
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Strategies to guide the affinity maturation process
Proposed immunization strategies III. Based on viral diversity Heterologous immunizations of well characterized molecules a mixture format Sequential immunizations Generation of somatic hypermutation (focus on specific site) Inundation of immunes system w epitopes of interest Vaccine concepts for induction of cross-reactive Ab II. Glycans not recognized by germline Ab but are neutralized by intermediate/mature Ab 11/13/2018 Unimi - MBC - Molecular Immunology
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Strategies to guide the affinity maturation process
Proposed immunization strategies Based on Ab ontogeny Based on Ab-viral co-evolution Based on viral diversity Based on epitope of interest Advantages of each strategy over the others (III.) Increase viral antigenic variation compared to using sequence of single donor (like in II.) Virus-Ab co-evolution data only available for a few epitopes Sequential immunogens more effective than mixed immunogens (Wang et al.) Choice of strategies Vaccine concepts for induction of cross-reactive Ab II. Glycans not recognized by germline Ab but are neutralized by intermediate/mature Ab 11/13/2018 Unimi - MBC - Molecular Immunology
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Strategies to guide the affinity maturation process
Help along the way Using vaccine platforms of sufficient immunogenicity Presence of antigen for extended periods of time Preclinical vaccines relied on non-replicating vectors or protein subunits extended Ag presence may be achieved w replication-component vectors non-replicating systems w slow release/ long residency Adjuvants may be crucial for affinity maturation Augmentation of T follicular helper cell interactions needs to be further developed 11/13/2018 Unimi - MBC - Molecular Immunology
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Strategies to guide the affinity maturation process
Determining our arrival Choosing vaccine concepts that succeed in human trials Requires thoughtful measurements of immunity Assessment of studies containing Immunogens Adjuvants Immunization schemes Animal models and resulting in conjunction w protection against challenge is critical Universal influenza/ HIV-1 vaccine Development of SHM Development of bnAb Development of cross-reactive Ab Sera neutralization assay combined w SHM by cloning Ag-specific B cell or by high-throughput next-generation sequencing SHM = somatic hypermutation bnAB = broadly neutralizing Antibody 11/13/2018 Unimi - MBC - Molecular Immunology
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Strategies to guide the affinity maturation process
Conclusion Effective immunization scheme Characterized and stable immunogens Containing bn-epitopes over non-neutralizing ones Priming immunogens Capable to bind to unmutated ancestors (bnAb families) Stimulation of immune system Generation of sustained response (over time) Maximize appropriate T helper functions Improvement of successful vaccination Requirements: Thoughtful choices of: the initial steps boosting immunogens Adjuvants Duration of the regimen (also well-defined and carefully measured goals) SHM = somatic hypermutation bnAB = broadly neutralizing Antibody 11/13/2018 Unimi - MBC - Molecular Immunology
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Strategies to guide the affinity maturation process
Questions SHM = somatic hypermutation bnAB = broadly neutralizing Antibody 11/13/2018 Unimi - MBC - Molecular Immunology
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Gaetano Cannavale & Lavinia Grasso
Milano, 11/13/2018
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Strategies to guide the affinity maturation process
Influenza virus variability Two strains of Influenza virus: Influenza A affects many avians and mammals Influenza B affects only humans High antigenic variability due to: Antigenic DRIFT accumulation of mutations in the antigens, resulting in changes of the epitope sequences Antigenic SHIFT combination among different viral strains to make a new one 11/13/2018 Unimi - MBC - Molecular Immunology
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Strategies to guide the affinity maturation process
Protection against Influenza virus Protection elicited by current seasonal influenza vaccines against viral deseases is antibody-mediated These antibodies bind to viral surface proteins that mediated virus entry or budding from host cells, leading to neutralization, opsonization and/or complement fixation 11/13/2018 Unimi - MBC - Molecular Immunology
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Strategies to guide the affinity maturation process
Challenges in eliciting desirable antibody responses against viral surface proteins Finding sequence-conserved and structure-conserved epitopes, that can elicit protection from diverse virus strains These proteins may exist in different forms and shapes, depending on wheter the viral particles are mature or immature, so some epitopes may be exposed in one form or shape, but not in others These proteins often go through conformational changes when they interact with their receptors or coreceptors or when they are endocytosed into the endosomal compartement These proteins often assemble into higher-order structures, so there can be different quaternary epitopes Some of these proteins contain specific glycans, that affect the antigenicity and immunogenicity of these epitopes 11/13/2018 Unimi - MBC - Molecular Immunology
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Strategies to guide the affinity maturation process
Three different vaccine design strategies REVERSE VACCINOLOGY analyze protein sequence diversity and then design ancestral, consensus, mosaic and multivalent immunogens SEROLOGIC VACCINOLOGY analyze cross-reactivity of antibody responses among subtypes or clades of a given virus and then design immunogens based on antigenic clusters ANALYTIC VACCINOLOGY analyze the human immune responses from infected or vaccinated individuals, isolate NAbs, determine the neutralization epitopes and then design immunogens based on epitope structures 11/13/2018 Unimi - MBC - Molecular Immunology
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Strategies to guide the affinity maturation process
Epitope-focused vaccine design against influenza hemagglutinin (HA) Viral surface protein Trimer of HA1-HA2 heterodimers HA1 allows virus to be endocytosed HA2 mediates viral-endosomal membrane fusion HEAD + STEM structure 18 subtypes (divided in group 1 and 2) in Influenza A Victoria and Yamagata antigenic lines in Influenza B 11/13/2018 Unimi - MBC - Molecular Immunology
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Strategies to guide the affinity maturation process
What we know During the past years, several monoclonal antibodies (mAbs) against the head and stem with various degrees of cross-reactivity have been isolated and their corresponding conserved epitopes have been elucidated Stem contains more conserved epitopes than head Head also contains some conserved epitopes present in diverse strains of different subtypes but their conservation appears more limited Head contains epitopes that are conserved in diverse strains within a HA subtype. These epitopes are located in the RBS or outside the RBS 11/13/2018 Unimi - MBC - Molecular Immunology
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Strategies to guide the affinity maturation process
What we know During the past years, several monoclonal antibodies (mAbs) against the head and stem with various degrees of cross-reactivity have been isolated and their corresponding conserved epitopes have been elucidated The antibody repertoire against epitopes in the head is much more diverse than the antibody repertoire against epitopes in the stem. Occlusive nature of the epitopes in the stem on virions: only a few antibodies with a specific mode of action can interact with these epitopes As a result, antibody responses against the head are more potent and dominant than those against the stem 11/13/2018 Unimi - MBC - Molecular Immunology
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Strategies to guide the affinity maturation process
STEM-BASED VACCINE DESIGN The discovery of broadly neutralizing mAbs against conserved epitopes in the stem has spurred great efforts on HA stem-based cross-subtype (‘universal’) vaccine design. Two approaches have been tested: Sequential cHA infections (chimeraHA with common stem + different subtypes head) Headless HA development 11/13/2018 Unimi - MBC - Molecular Immunology
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Strategies to guide the affinity maturation process
Sequential cHA infections Sequential prime-boosts with cHA containing a common H1 stem were able to cross-protect mice from lethal challenge of H5N1 and H6N1 viruses (group1) and to cross-neutralize H2N2 virus (group2) Sequential prime-boosts with cHA containing a common H3 stem were able to cross-protect mice from lethal challenge of H7N1 and H7N9 viruses (group2) 11/13/2018 Unimi - MBC - Molecular Immunology
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Strategies to guide the affinity maturation process
Headless HA development Earlier studies to remove HA head chemically or genetically resulted in limited success misfolding of the headless HA leading to concomitant loss of conserved conformational epitopes Impagliazzo et al. reported an H1HA stem that mantains conserved conformational epitopes. Mice ibridized with it showed cross-neutralization against H1 and H5 and many binding Abs Yassine et al. generated a self-asslebled nanoparticle by linking HA-SS with H. pylori ferritin. Mice and ferrets immunized with H1 HA-SS-np showed many binding Abs production and NAbs only against H1 11/13/2018 Unimi - MBC - Molecular Immunology
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Strategies to guide the affinity maturation process
Problems Although the successful design of stable, correctly folded headless HA is an important advancement towards ‘universal’ influenza vaccine, many questions remain Both constructs contains heterologous elements that can induce autoimmunity in humans Studies in swine models have been shown to promote virus fusion and enhance influenza virus respiratory disease Both constructs cross-protect heterosubtypic H5 challenge with no robust neutralizing antibody responses 11/13/2018 Unimi - MBC - Molecular Immunology
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Strategies to guide the affinity maturation process
RBS-BASED VACCINE DESIGN The HA head is less conserved than the stem and prone to antigenic drift, but the discovery of many anti-RBS antibodies that cross-neutralize diverse strains within subtypes H1, H3, H5 and H7 and within influenza B viruses has spurred efforts on RBS-based HA subtype-specific vaccine design Currently only H1 and H3 subtypes of influenza A viruses and both lineages of influenza B viruses circulate in human population (others are just occasionally transmitted by avians). Thus if RBS-based vaccines can elicit subtype-specific protective antibody responses against these subtypes, one may take care of antigenic drift problem 11/13/2018 Unimi - MBC - Molecular Immunology
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Strategies to guide the affinity maturation process
Self-assembling HA-ferritin Kanekiyo et al. designed self-assembling nanoparticles by genetically linking the ectodomain of HA from H1, H3 and Influenza B to H. pylori non-haem ferritin HA-ferritin fusion protein produced by a mammalian expression system spontaneously self-assembled into 24mer nanoparticles Immunogenicity studies indicate that this particle spreads a NAbs response against both the stem and the RBS on the head This lead, in ferrets, to the protection against all the H1 viruses from 1934 to 2007, solving the antigenic drift problem 11/13/2018 Unimi - MBC - Molecular Immunology
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Strategies to guide the affinity maturation process
Questions SHM = somatic hypermutation bnAB = broadly neutralizing Antibody 11/13/2018 Unimi - MBC - Molecular Immunology
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Strategies to guide the affinity maturation process
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Strategies to guide the affinity maturation process
Vaccination in children Children, especially young children, are more sensible than adults to both Complications related to diseases caused by influenza virus Rare serious adverse events to influenza vaccines The purpose of this review is to summarize the available published English-language literature on the safety of influenza vaccines in children to assist decision-making regarding recommendations for use of these vaccines in children 11/13/2018 Unimi - MBC - Molecular Immunology
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108 vaccines were identified Produced by 47 manufacturers in 27 different countries 98 vaccines by inactivated virus 10 vaccines by live attenuated virus The list of vaccines considered in the review includes information from the WHO (World Health Organization) table of pandemic and seasonal influenza vaccines accessed October 30, 2014 11/13/2018 Unimi - MBC - Molecular Immunology
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Adverse events were selected for review if there was evidence of possible causal relationships with one or more influenza vaccines They were included in: guidelines for the use of influenza vaccines; reviews by the IOM (Institute of Medicine); published reviews on influenza vaccines safety; a literature review for vaccine safety and influenza vaccines. PubMed and EMBASE databases were searched separately for each adverse event and category. 11/13/2018 Unimi - MBC - Molecular Immunology
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Criteria accepted as evidence of a causal Relationship Evidence of an increased risk in vaccines recipients as compared to controls Evidence of an increased biologically plausible time window as compared to control windows Local reactions on the sites of the injection when there is no evidence of other possible causes With live attenuated vaccines, evidence of the vaccines virus or bacterium confirmed by genetic sequencing in the affected tissue of patients with a serious adverse event Immediate hypersensitivity reactions occurring within 4h after immunization when there is no evidence of other possible exposures that could have resulted in a hypersensitivity reaction 11/13/2018 Unimi - MBC - Molecular Immunology
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Some adverse events that are causally associated with influenza vaccines occur only or primarly in adults, and some others occur more frequently only in children. A sufficient number of studies was conducted in adults, but not in children Whenever possible, data related to studies conducted in children are shown In instances, where the data on safety have been generated primarly in adults, an assessment of the likelihood that the adult data are applicable to pediatric population was provided 11/13/2018 Unimi - MBC - Molecular Immunology
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Unimi - MBC - Molecular Immunology
Adverse events analyzed Local reactions following IIV Cellulitis-like reactions Hypersensitivity reactions Fever and febrile seizures Malaise, myalgia, and related symptoms Inflammatory arthritis Guillain-Barré syndrome 11/13/2018 Unimi - MBC - Molecular Immunology
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Adverse events analyzed Acute Disseminated Encephalomyelitis (AEDEM), Multiple Sclerosis (MS) and transverse myelitis (TM) Narcolepsy Bell’s palsy Immune thrombocytopenia (ITP) Adverse events following LAIV (like fever and wheezing) 11/13/2018 Unimi - MBC - Molecular Immunology
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Results Most influenza vaccines are generally safe Rare serious adverse events (6001 published articles as reference) Different safety in different populations due to different genetic predisposition Risk of adverse events Lower than virus diseases 11/13/2018 Unimi - MBC - Molecular Immunology
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Strategies to guide the affinity maturation process
Thanks for your attention Lavinia, Zeynep, Gaetano and Manuel SHM = somatic hypermutation bnAB = broadly neutralizing Antibody 11/13/2018 Unimi - MBC - Molecular Immunology
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