Mastering Immunity Europe 2019 speakers announced and registration open
Mastering Immunity Europe 2019 will be taking place at Wolfson College, University of Oxford on 21 March. Speakers have now been announced and registration is now open.
MHC Pentamers used by team at GSK to study mRNA vaccines that can protect against several Flu strains
used by team at GSK to study mRNA vaccines that can protect against several Flu strains
Magini D. et al., PLOS One (2017)
mRNA Vaccines Expressing Multiple Conserved Influenza Antigens
Confer Protection against Homologous and Heterosubtypic Viral
CD8+ T-cell responses in lungs after influenza
challenge. BALB/c mice
were immunized i.m. twice, 8 weeks apart, with PBS, 0.1 μg of
self-amplifying mRNA (SAM®) vectors SAM(NP), SAM(M1),
SAM(M1-NP), or with 0.2 μg of SAM(NP)+SAM(M1). Four weeks after
the second immunization, mice were infected with PR8 virus.
NP-specific CD8 T cells recruited in the lungs after the
infection were characterized by flow cytometry. (a) Numbers of
NP-specific CD8+ T cells determined using
Pro5® MHC pentamers. Data
are from individual mice (depicted as dots), while solid lanes
indicate the mean±SD. (b) Cumulative frequency of Ag-specific,
cytokine-secreting CD8+ T cells, indicated as
absolute number per lung. The color code represents the
different combinations of cytokine produced by the respective
cells after in vitro stimulation with medium (m), NP147-155
peptide (NP), or M1 peptide pool (M1), as indicated. (c)
Absolute number of NP-specific CD8+ T cells positive
(black bar) or not (grey bar) for CD107a. Data derived from two
independent and merged experiments. Statistical analyses were
performed using the Mann-Whitney U test. *p<0.05; **p<0.01
compared to the PBS-treated group.
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Current hemagglutinin (HA)-based seasonal influenza vaccines induce
vaccine strain-specific neutralizing antibodies that usually fail to
protect against mismatched circulating viruses. Inclusion of
conserved proteins such as the nucleoprotein (NP) and the matrix
protein 1 (M1) can increase effectiveness by eliciting
cross-reactive T-cells. However, efficient priming of T-cell
responses requires the right delivery system. Here we show novel
self-amplifying mRNA (SAM®) vectors expressing influenza
NP (SAM(NP)), M1 (SAM(M1)), and NP and M1 (SAM(M1-NP)) delivered
with lipid nanoparticles (LNP) induce robust polyfunctional CD4 T
helper 1 cells, while NP-containing SAM also induced cytotoxic CD8 T
cells. Robust expansions of central memory (TCM) and
effector memory (TEM) CD4 and CD8 T cells were also
measured. An enhanced recruitment of NP-specific cytotoxic CD8
T cells was observed in the lungs of SAM(NP)-immunized mice after
influenza infection that paralleled with reduced lung viral titers
and pathology, and increased survival after homologous and
heterosubtypic influenza challenge. We show for the first time that
the co-administration of RNA (SAM(M1-NP)) and protein (monovalent
inactivated influenza vaccine (MIIV)) was feasible, induced
simultaneously NP-, M1- and HA-specific T cells and HA-specific
neutralizing antibodies, and enhanced MIIV efficacy against a
Pro5® MHC Pentamer
Pro5® MHC Class I Pentamers are the market leading product for detecting antigen-specific T cells, you can count on consistent performance backed up by unrivalled customer service.
To ensure uniformity and consistency, each Pentamer is purified four times before being subjected to rigorous quality control acceptance testing.
USA & Canada, Toll Free: +1 888 505 7765
All other countries: +44 (0)870 042 7279
More on Pro5® Class I MHC Pentamers
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ProImmune launches MutaMap™ Mutational Activity Map Service
When it’s time to make final engineering decisions for your antibody or protein, MutaMap™ can help evaluate which individual point mutations to pursue. MutaMap™ is an in vitro assay system that helps explore the effect of substituting each amino acid in at each position in a protein sequence one by one with all 19 possible substitutions and find out the effect on protein activity. For example a sequence stretch of 100 amino acids will result in up to 2000 mutants to explore.
The approach of MutaMap™ is simple.
Each position of a protein of interest is mutated by site directed mutagenesis, expressed and tested for its affinity/activity. MutaMap™ does not use any surrogate measurement for affinity or activity. Cell free in vitro translation of proteins is combined with solution titration assays to measure affinity/activity. Both methods are optimized for high throughput processing of samples while still allowing for accurate measurements of affinity/activity. The technology is particularly suitable for investing the ligand binding interactions of high affinity monoclonal antibodies, down into the high femtomolar range where other approaches such as SPR struggle to deliver high
Watch our MutaMap™ Podcast
Mutations investigated in CDR1 and CDR3 of Avastin® heavy chain variable domain
Shown above is an example of a mutagenesis heatmap generated for CDR1 and CDR3 from Avastin® heavy chain variable fragment.
For binding pair interactions MutaMap™ uses high throughput solution equilibrium titration (SET) immunoassays to determine the binding affinity for each construct tested.
SET results for measuring the affinity of Avastin® scFv and
Lucentis® scFv. The resulting titration curve is regressed
according to the relevant mass action binding laws. The
robustness of the SET approach ensures that this high throughput
assay works well for affinities in
the single digit picomolar range, as demonstrated by the tight confidence
What does MutaMap™ show you?
MutaMap™ delivers a heat map for your protein (see Figure 1 above) that shows you which point
mutations lead to an increase, decrease, or no change in affinity (or
other activity) or non-function of the protein when interacting with one
or more of its binding partners. Effectively you can learn which
mutations, one by one, are likely to be permissible or favourable in your
protein in terms of the key property of binding to a ligand.
MutaMap™ therefore allows you to make informed protein
engineering decisions for a range of key developability objectives which
Focus on position S105 in CDR-H3 of Avastin®
heavy chain variable domain
Figure 3: What is clear is that the MutaMap™ heatmap shows permissible
mutations, especially in CDR-H3 in cases that are not normally
considered conservative, e.g. in position S105. This opens up choices for re-designing the molecule
that would not normally be available based on computational assessments.
Figure 4: Position S105 in Avastin® CDR-H3 is mutated to T in
in Lucentis® MutaMap™ reconfirms that this mutation is indeed beneficial for
improving binding. It also shows that a number of other mutations
are available to match or improve the affinity of the construct
over the Avastin® wild type.
How does MutaMap™ compare to molecular evolution technologies?
Molecular evolution techniques such as phage display and other phenotype-genotype
coupled randomization techniques are most commonly used in the affinity
maturation process for monoclonal antibodies and other binding
scaffolds. The advantage of these technologies is that they help explore
a very large sequence space of combined mutations.
There comes a point
however when final decisions have to be made on the implementation of a
protein sequence where individual point mutations may be considered in
an antibody or therapeutic protein to meet a variety of design
objectives. Randomized molecular evolution is not appropriate for this
step. Exploring individual point mutations is nothing new, but it has
been difficult to carry this step out in very high throughput way,
especially where the objective is to clone and express every mutant and
then measure its affinity/activity with reasonable accuracy. This
is what MutaMap™ can achieve.
Where does MutaMap™ fit in as part a project for e.g. generating a candidate
monoclonal antibody for clinical development?
Figure 5:Example work flow for pre-clinial protein engineering of therapeutic
monoclonal antibody; individual projects may differ.
How long does a MutaMap™ project take?
Our objective is to complete medium size projects of exploring 500-2000 mutations in
approximately 8-12 weeks from receiving the customer’s protein sequence.
Larger projects will take slightly longer, depending on complexity.
What is the stepwise process for carrying out MutaMap™?
we will discuss with you the sequence space you want to explore. This
may be the known paratope of a protein or the CDR and flanking regions
of a monoclonal antibody.
To save time and cost you may not want to explore substituting amino
acids into the sequence that are considered highly non-conservative or
prone to degradation when exposed. We will also discuss what you know
about the binding interaction of your protein with its target, whether
you have working immunoassays for this interaction and whether the wild
type protein and isolated ligand is available to work with in an
immunoassay system. Depending on the nature of the interaction and the
binding partners we will ensure that the base assay of binding wild type
protein to target works well.
details are agreed and the project is initiated we will run the
mutagenesis agreed for each position, express the protein at small scale
and carry out the equilibrium binding titration affinity measurement in
What will you get?
A final technical
report delivered via our secure webserver showing you the affinity or
activity determination for the wild type and each mutant with confidence
interval. This will be presented in various formats for ease of
interpretation, including a standard heatmap.
For customers that
want to carry out protein antigenicity studies in parallel, these can be
carried out in approximately the same timeframe as the MutaMap™. This
means that within a period of approximately 8-12 weeks we will have
determined experimentally both the putative T cell epitopes and the
MutaMap™ of permitted mutations in your protein sequence. This
information can allow you to proceed with much better informed decisions
on how to address immunogenicity related issues for your program while
addressing simultaneously other developability related design decisions
for your sequence.
Introducing: ProT2™ MHC Class II Tetramers
Oxford, UK, 14 January 2016
ProImmune, a leader in services for understanding and managing adaptive
immune responses, announced today the introduction of ProT2™,
a range of Human MHC Class II tetramer reagents for tetramer analyses in
the study of antigen-specific CD4+ T cell immune responses. ProImmune’s
offering represents the broadest choice of ready to order catalog and
custom items in this field and will enable research into CD4+ T cell
responses in a variety of disease areas including cancer, infectious
diseases, allergy, autoimmunity and transplantation. Another key area
for this product range is the detailed study of T cell help in unwanted
immune responses to biologics.
CD4+ T cell
responses are at the heart of steering the adaptive immune system
directing both cytotoxic T cell responses and antibody responses
mediated by B cells. ProT2™ MHC Class II Tetramers allow the direct
detection and separation of single antigen-specific CD4+T cells
accurately by flow cytometry.
Schwabe, CEO of ProImmune commented: “By introducing a broad and growing
ProT2™ Tetramer product range we intend to give MHC Class II tetramer
analysis the emphasis it deserves in the study of CD4+ T cell responses
which play such a pivotal role in adaptive immunity across so many
disease and treatment modalities.”
Class II MHC Monomer reagents are supported by ProImmune’s expert
customer service and application support, helping new users in
particular to establish and optimize robust protocols in their own
addition to ProImmune’s class II reagent offering further complements an
already extensive range of services for evaluating CD4+ T cell
responses, including ProImmune REVEAL® MHC peptide binding assays, CD4+
T cell and DC T cell proliferation assays and standard immune monitoring
services through ELISPOT and flow cytometry based assays.
information on ProT2™ reagents and their availability, or to place an
order, please visit:
, or call + 1 888 505 7765 in the United States or + 44 870 042 7279 in other countries.
Notes to Editors:
About ProImmune –
ProImmune’s mission is to
be a partner of choice for understanding and managing adaptive immune
responses. It does this by offering unique solutions for preclinical and
clinical immunology research, including a comprehensive and integrated
antigen characterization and immunogenicity testing platform, and
products and services for tracking antigen-specific immune responses
with state-of-the-art ELISpot and flow cytometry techniques. ProImmune
is committed to helping researcher’s achieve success through product
innovation, responsive service and focused application support, saving
time and money, and reducing risk.
For more information, please contact
Tel: +44 (0) 870 042 7279
Zyme Communications Ltd
Tel: +44 (0) 7771 730 919
ProImmune Introduces ProSentium™ a Unique Peptide Sequence Database to Investigate T Cell Immune System
Oxford,UK, 14 April 2015
– ProImmune, a leader in services for understanding and managing
adaptive immune responses, announced today the introduction of a new
antigen database service called ProSentium™, based on
assay results using sequencing mass spectrometry. ProSentium data
can show the precise peptide sequences from proteins of interest
visible to the body’s own T cell immune system. The data
collection covers many high value therapeutic targets and areas including the
majority of licensed replacement factor proteins, including clotting
factors, replacement enzymes, spanning development areas including
oncology, cardiovascular, CNS, immunology, gastroenterology and many
Under an exclusive or non-exclusive license, the bespoke database service
investigates and reports peptide hits against the ProSentium
database for a customer’s specific protein or protein family of
interest. Results are reported as overlapping nested peptide sets
and provide statistically relevant antigen sequence data that can be
used to further inform drug development decisions.
CEO of ProImmune commented: “ProSentium supports pivotal decision
making in drug design and development with the physical evidence
required for game changing approaches that target or interact with
the immune system. Through our service offering, we are delighted
that we can offer this ground-breaking resource to researchers so
they can better understand the body’s immune response and bring new
therapies to patients. ”
T cells play a
critical role in all immune responses, including in fighting
infections, cancer and in autoimmunity and in unwanted drug
reactions. To understand T cell responses, it is crucial to
understand the specific peptide antigens that the immune system
recognizes and are presented on the cell surface of both normal and
diseased cells. ProSentium data can be readily analyzed and
reported, and is fully compatible with ProImmune’s in vitro
assay services which can be combined to help investigators gain a
more complete understanding of relevant immune responses at a
For more information on ProSentium please visit:
, or call +1 888 505 7765 in the United States or +44 870 042 7279 inother countries.
More on ProSentium™
About ProImmune –
ProImmune’s mission is
to be a partner of choice for understanding and managing immune
responses. It does this by offering unique solutions for preclinical
and clinical immunology research, including a comprehensive and
integrated antigen characterization and immunogenicity testing
platform, and products and services for tracking specific immune
responses. ProImmune is committed to helping researcher’s achieve
success through product innovation, responsive service and focused
application support, saving time and money, and reducing risk.
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