1. Strategies to guide the affinity maturation process
Zeynep Tellioğlu & Manuel Kenzler
Milano, 13/11/2018

2. 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
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3. 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
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4. 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.
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5. 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
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6. 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
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7. 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
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8. 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
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9. 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
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10. 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
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11. 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
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12. 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.
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13. 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
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14. 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
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15. 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
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16. 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
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17. 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
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18. 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
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19. 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
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20. 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
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21. 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
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22. 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
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23. Strategies to guide the affinity maturation process
Questions
SHM = somatic hypermutation
bnAB = broadly neutralizing Antibody
11/13/2018
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24. Gaetano Cannavale & Lavinia Grasso
Milano, 11/13/2018

25. 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
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26. 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
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27. 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
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28. 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
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29. 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
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30. 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
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31. 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
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32. 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
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33. 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
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34. 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
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35. 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
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36. 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
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37. 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
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38. Strategies to guide the affinity maturation process
Questions
SHM = somatic hypermutation
bnAB = broadly neutralizing Antibody
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39. Strategies to guide the affinity maturation process

40. 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
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41. Unimi - MBC - Molecular Immunology
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
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42. Unimi - MBC - Molecular Immunology
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.
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43. Unimi - MBC - Molecular Immunology
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
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44. Unimi - MBC - Molecular Immunology
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
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45. 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
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46. Unimi - MBC - Molecular Immunology
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)
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47. Unimi - MBC - Molecular Immunology
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
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48. 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