structural vaccinology, in which immunogens are rationally engineered using available structural information.

2. Structural Vaccinology
By: Nasim Arshadi
Shahed university,
Department of biology
2017

3. History
Structural vaccinology definition
The need for structural vaccinology
Structural vaccinology classification
Conclusion
3

4. During the last 30 years, several new technologies made
possible vaccines that were previously impossible
4

5. Today we see an explosion of new technologies
2014
5

6.  In terms of identifying appropriate antigens and/or
engineering antigens to obtain optimal vaccine
responses, considerable work has focused on two
broad areas:
1. reverse vaccinology, in which novel immunogens
are discovered via whole genome bioinformatics
2. structural vaccinology, in which immunogens are
rationally engineered using available structural
information.
6

7. 7
Majoraimsin SV are the identification of protective Bcell epitopes on
the antigens and optimizing the antigens in terms of stability, epitope
presentation, ease of production and safety.
antigen engineering has the potential to optimize not only the immunogenicity,but also
the stability and the industrial production of pro-mising vaccine candidates

8. Structural vaccinology
8
 Efficacious immune response does not require recognition of the
entire antigenic protein, but that recognition of a single or multiple
selected epitopes may be sufficient to induce protective
immunity.

9.  Experimental method , X-ray crystallography, Nuclear
Magnetic Resonance (NMR) spectroscopy and more recently
Transmission Electron Microscopy (TEM), are the most used
techniques for protein structure determination, combined with
computational methods, as computational scaffold design and
epitope prediction are intensively used as alternative tools to
predict the three-dimensional structures and design of B-cell
epitopes.
9

10. The need for structural vaccinology

11. We will review recent studies in structure-based
vaccine design, in which immunogens present one
or more key epitopes or immunogenic domains,
with the goal of inducing epitope-specific antibodies
and/or yielding a broad coverage antibody response.
1. Broad-coverage immunogens
2. Epitope-focused immunogens
3.Germline-targeting immunogens
11

12. Broad-coverage immunogens
Vaccines are often required to elicit protective
responses against diverse strains of a pathogen.
The group B serotype of Neisseria meningitis (MenB) is a
bacterial pathogen without a vaccine, due in part to
the high antigenic variation.
12

13. Neisseria meningitis
13

14. In silico vaccine
candidates
Express
recombinant
proteins
VACCINE CANDIDATES
600 potential vaccine candidates identified
350 proteins successfully expressed
in E.coli
91 novel surface-exposed
proteins identified
28 novel proteins
have bactericidal
activity
Reverse Vaccinology
Agenomic approach to vaccine discovery

15. Factor H Binding Protein (FHBP)
fHbp is a 27 kDa surface lipoprotein consisting of two ßbarrels.
divided into three variant groups, V1, V2,and V3 on its predicted
amino acid sequence.
allows meningococcus to escape complement-mediated killing by
the immune system.
fHbp is very effective at eliciting protective antibodies, but it has more
than 500 known amino acid sequence variants
fHbps belonging to the same variant group share over 85% amino
acid identity, and only 60–70% similarity between variant group.
immunological cross-reactivity within, but not between, variant groups
 To induce strong protective immunity against all three variants,
antigens specific for each variant must be included in the vaccine,
which makes the manufacturing process complex and expensive.
15

16. amino acids important for recognition by the antibodies against variant 1
colored red
amino acids important for recognition of variants 2 and 3 colored purple
16
The protective epitopes of variant 1 and of variants 2 and 3
map in nonoverlapping regions located mostly in the amino-
and carboxy-terminal regions of fHbp, respectively.

17.  Variant 1 was used as a lead molecule, a scaffold, and regions
containing specific residues from epitope of variant 2 and variant
3 were introduced in various regions of variant 1.
As the C-terminal domain contains the majority of
protective epitopes, in order to determine in a fine-tune way
the protective residues,
The C-terminal region of variant 1 was divided into ten areas
large enough to contain at least one conformational epitope
(approximately 900–2,000 Å (
the residues of the variant 1 C terminus were then replaced with
the corresponding amino acids of variants 2 and/or 3, regardless
of their position in the primary sequence.
To preserve folding, amino acid substitutions were introduced
only for residues with side chains that are well exposed to
solvent, leaving the internal core of the protein unaltered.
17

18. 18

19. Biochemical characterization of selected candidates
• To verify that the mutations introduced did not cause major
alterations in protein folding or stability, we analyzed the
selected mutants
• to evaluate different biochemical and biophysical properties
when compared with the serobase fHBP subvariant 1.1.
• In particular, the tendency to aggregate, secondary
structure, and folding were monitored by size exclusion
high-pressure liquid chromatography (SEC-HPLC), circular
dichroism (CD) spectroscopy, and NMR, respectively.
• Comparison with retention times of reference molecules
indicated that the mutants were present as monodisperse
monomers and did not form aggregates.
19

20. G1 elicited titers >1000 against most of the strains tested. Arg
204 ,originally described as part of a bactericidal epitope of
variant 1, was mutated in G1 to serine without destroying
variant 1 immunogenicity. This suggested that it was the larger
surface area and not the individual amino acids that was
important for immunogenicity.
To further optimize the immunogenicity, Thus, two copies of G1
were fused to GNA2091, a meningococcal antigen that
enhances the stability and efficacy of fHBP variant 1, although
it does not itself induce bactericidal antibodies.
20

21. Epitope-focused immunogens
21

22. Group B Streptococci
Gram-positive pathogens
commonly found in the uro-genital tract, with about 25% of women
carrying it at any time, often having no symptoms.
Pregnant women with group B strep infection or colonization can transmit
the bacteria to the baby during delivery GBS transmission in infants can
cause life-threatening infections in newborns in the hours after birth,
during the first week of life, causing the early onset disease (EOD) or
even several months later, late onset disease (LOD) cause serious
illness in infants and the elderly.
22

23. Cell-surface pili :have direct roles in
virulence and also serve as protective
antigens.
reverse vaccinology approach applied to
the 8 sequenced genomes of GBS.
However, for several reasons, an effective
anti-GBS vaccine is not yet available.
One reason :there are ten GBS serotypes,
necessitating a complex vaccine if serotype-
specific immunogens are selected.
It was recently shown that all GBS strains
express pili, which are long filamentous
structures involved in bacterium–host
interactions, bacterial aggregation and
biofilm formation
23

24. Comparative analysis on the complete genome
sequences available for GBS revealed three independent
loci named Pilus Islands, PI-1, PI-2a and PI-2b encoding
structurally distinct pilus types. each GBS strain can carry
one or two Islands.
GBS pili are composed of three structural proteins,
1) backbone protein (BP), which forms the pilus shaft;
2) ancillary protein 1 (AP1), which decorates the pilus
stem;
3)AP2, which is often found at the base of the pilus and
anchors it to the cell wall.
 All three proteins are covalently linked to each other
through a sortase-mediated transpeptidation reaction.
24

25. DNA sequence analysis has shown that the three subunits in
strains carrying the same island are highly conserved, with the
exception of BP-2a, which is grouped into six main different
immunologically variants.
Furthermore, the BP encoded by PI-2a (BP-2a) has six
sequence variants
The 3D structure of one of the six BP-2a variants suggested a
possible solution, as it revealed a four-domain organization in
which domain three (D3), which is 100 amino acids long, is
likely to face the external side of the pilus shaft, based on the
capacity of D3-specific antibodies to bind GBS.
D3 elicits high titres of opsonophagocytic antibodies, which
protect mice against lethal challenge with GBS isolates
expressing the PI-2a pilus.
25

26. Because of their small size, the D3 domains from each
variant could be fused into a single recombinant construct
that is efficiently expressed in Escherichia coli and can be
purified.
The recombinant chimaera confers strong protection
against all strains expressing a BP-2a variant.
This structure-based work might pave the way for the
development of a universal, broadly protective GBS
vaccine.
In more general terms, this work revealed that specific
structural domains within a protein can sometimes be
sufficient to elicit a protective immune response
26

27. 27

28. 28

29. Germline-targeting immunogens
The high antigenic diversity of viruses such as HIV-1,
influenza virus, and hepatitis C virus poses major
challenges for vaccine design because large portions of
the surfaces of envelope glycoproteins are variable and
covered by glycans
29

30. • The discovery of broadly neutralizing antibodies (bNAbs) that
neutralize diverse strains of HIV,influenza, or HCV gave rise to
a strategy in which a bNAb is employed to guide design of
immunogens to induce responses similar in structural recognition,
breadth and potency.
30
Env
gp120
the CD4
receptor
binding site
hyper-variable
loops.
gp41
Playing a critical role in virus infection
and pathogenesis.

31. • The VRC01 antibody is able to bind onto HIV at the CD4
binding site on the gp120 protein. This neutralizes HIV
and prevents HIV from being able to attach to cells and
infect them.
• Such broadly neutralizing antibodies typically work by
blocking crucial functional sites on a virus that are
conserved among different strains despite high mutation
elsewhere.
31

32. • These bNAbs are highly mutated from germline, and have been
produced by HIV-infected individuals only after two to three years of
infection.
• Hence it is expected that elicitation of similar bNAbs by vaccination
will be very difficult and may require a lengthy and complex
immunization regimen.
32

33. to elicit broadly neutralizing antibodies called VRC01
Germline B cells are major targets of modern viral vaccines,
because it is the initial stimulation of these B cells and their
antibodies that leads to a long-term antibody response.
you could try using the HIV envelope protein as your
immunogen
“but envelope protein doesn’t bind with any detectable affinity
to the B cells needed to launch a broadly neutralizing antibody
response.”
The team thus set out to design an artificial immunogen that
would be successful at achieving this.
33

34. The scientific team that has unveiled a new technique for vaccine
design includes Jean-Philippe Julien, Bill Schief, Joe Jardine and
Sergey Menis (left to right). (Photo by Cindy Brauer.)
34

35. 35

36. used a protein modeling software suite called Rosetta to
improve the binding of VRC01 germline B cell antibodies to
HIV’s envelope protein
We asked Rosetta to look for mutations on the side of the HIV
envelope protein that would help it bind tightly to our germline
antibodies
Jardine then generated libraries that contained all possible
combinations of beneficial mutations, resulting in millions of
mutants, and screened them using techniques called yeast
surface display and FACS.
This combination of computational prediction and directed
evolution successfully produced a few mutant envelope
proteins with high affinity for germline VRC01-class antibodies
36

37. Mimicking a Virus
Vaccine researchers know that such an immunogen typically
does better at stimulating an antibody response when it is
presented not as a single copy but in a closely spaced cluster
of multiple copies, and with only its antibody-binding end
exposed. “We wanted it to look like a virus
Menis therefore devised a tiny virus-mimicking particle made
from 60 copies of an obscure bacterial enzyme and coated it
with 60 copies of eOD-GT6. The particle worked well at
activating VRC01 germline B cells and even mature B cells in
the lab dish, whereas single-copy eOD-GT6 did not.
“Essentially it’s a self-assembling nanoparticle that presents
the immunogen in a properly oriented way,” Menis said. “We’re
hoping that this approach can be used not just for an HIV
vaccine but for many other vaccines, too.”
37

38. 38
engineered outer domain (eOD) of gp120

39. 39

40. conclusion
 The combination of structural biology and Reverse Vaccinology
has led to the evolution of Structural Vaccinology.
 One of the main strengths of SV is that atomic-level resolution
information can be used to rationally engineer the antigens,
thus considerably reducing the trial and error approach,
focusing efforts and reducing project timeline.
In light of these recent successes, and with an appreciation of
the aforementioned obstacles to antigen design, we anticipate
that SV will play an increasingly important role in the
development of future vaccines.
• Structure-based vaccines with reduced complexity and broad
efficacy could greatly enhance the number of people who might
benefit from the therapies that are developed.
40

41. Reference1.Cozzi R, Scarselli M, Ferlenghi I, Ferlenghi I. Structural vaccinology: a
three-dimensional view for vaccine development. Current topics in medicinal
chemistry. 2013;13(20):2629-37.
2.Dormitzer PR, Grandi G, Rappuoli R. Structural vaccinology starts to
deliver. Nature Reviews Microbiology. 2012;10(12):807-13.
3.Dormitzer PR, Ulmer JB, Rappuoli R. Structure-based antigen design: a
strategy for next generation vaccines. Trends in biotechnology.
2008;26(12):659-67.
4.Kulp DW, Schief WR. Advances in structure-based vaccine design. Current
opinion in virology. 2013;3(3):322-31.
5.Liljeroos L, Malito E, Ferlenghi I, Bottomley MJ. Structural and
computational biology in the design of immunogenic vaccine antigens.
Journal of immunology research. 2015;2015.
6.Nuccitelli A, Cozzi R, Gourlay LJ, Donnarumma D, Necchi F, Norais N, et
al. Structure-based approach to rationally design a chimeric protein for an
effective vaccine against Group B Streptococcus infections. Proceedings of
the National Academy of Sciences. 2011;108(25):10278-83.
7.Scarselli M, Aricò B, Brunelli B, Savino S, Di Marcello F, Palumbo E, et
al. Rational design of a meningococcal antigen inducing broad protective
immunity. Science translational medicine. 2011;3(91):91ra62-91ra62.
8.Seib KL, Scarselli M, Comanducci M, Toneatto D, Masignani V. Neisseria
41

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