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Proof of concept for a single-dose Group B Streptococcus vaccine based on capsular polysaccharide conjugated to Qβ virus-like particles


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A maternal vaccine to protect neonates against Group B Streptococcus invasive infection is an unmet medical need. Such a vaccine should ideally be offered during the third trimester of pregnancy and induce strong immune responses after a single dose to maximize the time for placental transfer of protective antibodies. A key target antigen is the capsular polysaccharide, an anti-phagocytic virulence factor that elicits protective antibodies when conjugated to carrier proteins. The most prevalent polysaccharide serotypes conjugated to tetanus or diphtheria toxoids have been tested in humans as monovalent and multivalent formulations, showing excellent safety profiles and immunogenicity. However, responses were suboptimal in unprimed individuals after a single shot, the ideal schedule for vaccination during the third trimester of pregnancy. In the present study, we obtained and optimized self-assembling virus-like particles conjugated to Group B Streptococcus capsular polysaccharides. The resulting glyco-nanoparticles elicited strong immune responses in mice already after one immunization, providing pre-clinical proof of concept for a single-dose vaccine.
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Proof of concept for a single-dose Group B Streptococcus
vaccine based on capsular polysaccharide conjugated to Qβ
virus-like particles
Filippo Carboni
, Roberta Cozzi
, Giacomo Romagnoli
, Giovanna Tuscano
, Cristiana Balocchi
, Giada Buf
, Margherita Bodini
Cecilia Brettoni
, Fabiola Giusti
, Sara Marchi
, Giulia Brogioni
, Barbara Brogioni
, Paolo Cinelli
, Luigia Cappelli
, Chiara Nocciolini
Silvia Senesi
, Claudia Facciotti
, Elisabetta Frigimelica
, Monica Fabbrini
, Daniela Stranges
, Silvana Savino
, Domenico Maione
Benjamin Wizel
, Immaculada Margarit
and Maria Rosaria Romano
A maternal vaccine to protect neonates against Group B Streptococcus invasive infection is an unmet medical need. Such a vaccine
should ideally be offered during the third trimester of pregnancy and induce strong immune responses after a single dose to
maximize the time for placental transfer of protective antibodies. A key target antigen is the capsular polysaccharide, an anti-
phagocytic virulence factor that elicits protective antibodies when conjugated to carrier proteins. The most prevalent
polysaccharide serotypes conjugated to tetanus or diphtheria toxoids have been tested in humans as monovalent and multivalent
formulations, showing excellent safety proles and immunogenicity. However, responses were suboptimal in unprimed individuals
after a single shot, the ideal schedule for vaccination during the third trimester of pregnancy. In the present study, we obtained and
optimized self-assembling virus-like particles conjugated to Group B Streptococcus capsular polysaccharides. The resulting glyco-
nanoparticles elicited strong immune responses in mice already after one immunization, providing pre-clinical proof of concept for
a single-dose vaccine.
npj Vaccines (2023) 8:152 ;
Streptococcus agalactiae (Group B Streptococcus, GBS) has adapted
to human entero-genital environments and colonizes 1530% of
. During pregnancy, these bacteria can reach the fetus
and cause stillbirth or preterm delivery
. Both pre-partum and
peripartum infections can result in severe neonatal bacteremia,
sepsis, and/or meningitis
. The risk of Early-Onset GBS disease in
the rst week of life has been reduced by intrapartum antibiotic
. However, this treatment does not protect against
GBS acquisition by the fetus during pregnancy or Late-Onset GBS
infection occurring between the second week and rst three
, and it is difcult to implement in poor-resourced
. A maternal vaccine against neonatal GBS infection is
therefore an urgent medical need.
GBS is surrounded by a thick sialic-acid-rich capsular poly-
saccharide (PS), which is a major virulence factor comprising ten
capsular PS serotypes
. Each PS when conjugated to carrier
proteins and administered to female mice prior to mating, elicited
serotype-specic functional antibodies that were efciently
transferred to pups and mediated their protection against GBS
lethal challenge
. In humans, PS serotype-specic maternal
antibody titers inversely correlated with the risk for infection in
, providing the basis to develop a PS-based maternal
vaccine for protecting neonates
. Interestingly, anti-PS IgG titers
in mouse and human sera strongly correlated with opsonopha-
gocytic killing titers, as well as with the levels of protection against
GBS infection in a passive immunization-neonatal challenge
mouse model
Maternal vaccines are offered during the third trimester of
pregnancy to maximize their safety, which leaves a short temporal
window to administer multiple doses. Therefore, a single-dose
formulation would be optimal for this population. GBS vaccines
containing one or multiple PS serotypes conjugated to Cross
Reactive Material (CRM, a non-toxic mutant of Diphtheria Toxin) or
Tetanus Toxoid (TT) have demonstrated to be safe in human
. However, one challenge encountered was the wide
range of measured anti-PS antibody titers after administration of a
single dose of vaccine. Indeed, while PS-specic IgG responses
were very high in individuals with pre-immune titers above the
Limit of Quantication (LOQ), and therefore presumably primed
by GBS colonization, those presenting baseline IgG titers below
the LOQ showed much lower responses. Antibody responses in
these unprimed subjects could not be improved using adjuvanted
formulations or by two vaccine doses administered after one or
two month intervals
, while a second dose offered more than 1
year apart strongly boosted PS-specic responses
. These
observations suggested that GBS PS conjugates were ideal for
boosting but might be sub-optimal priming vaccines. A need for a
second dose has also been observed for vaccines targeting GBS
proteins both in preclinical models
and in humans
In the present study we investigated the potential of an
alternative vaccine presentation by conjugating PS to Protein
Nanoparticles (NPs) and Virus-Like Particles (VLPs). NPs and VLPs
can be potent delivery systems for protein antigens due to their
large size and dense antigen display that enhance immune-cell
uptake and activation
. Several vaccines based on NPs and VLPs
have been developed for delivery of viral peptide and protein
NPs and VLPs are also receiving growing interest as carriers of
polypeptide antigens for developing vaccines against bacterial
. However, they have been less studied as carriers
GSK, Siena, Italy.
GSK, Rockville, MD, USA. email:;
Published in partnership with the Sealy Institute for Vaccine Sciences
of saccharide antigens
. Here we compared different self-
assembling NPs and VLPs as scaffolds for GBS PS antigens to
improve their immunogenicity after a single vaccine dose.
A single dose of GBS PSII conjugated to QβVLPs elicits IgG
and functional antibody responses comparable to two doses
of PSII-CRM conjugates
Similar to humans who are unprimed and present a low immune
response to GBS PS conjugated to proteins (TT or CRM), at least
two booster doses are required to elicit peak IgG responses in
mice, rabbits
, and non-human primates
. Therefore, we
assessed PS conjugated to self-assembling NPs and VLPs for
possible enhanced immune responses in a mouse immunization
model. The type II capsular polysaccharide (PSII), one of the six
most abundant serotypes (Ia, Ib, II, III, IV, and V), was conjugated to
NPs varying in subunit number and size, or to CRM as a
benchmark. The self-assembling NPs Ferritin
and mI3
as well
as bacteriophage Qβcoat protein VLP, with diameters of 10, 18,
and 30 nanometers, respectively, were selected and expressed as
recombinant proteins from genetically engineered E. coli. These
nanoparticle carriers and CRM were conjugated to PSII, after
random partial oxidation with sodium periodate, via reductive
amination of ε-amine groups of lysines
. The resulting PSII
conjugates were puried by Tangential Flow Filtration and
characterized for protein and saccharide content and tested for
immunogenicity in mice. The integrity of the nanoparticles before
and after conjugation was conrmed by size-exclusion High
Performance Liquid Chromatography (HPLC), Differential Light
Scattering (DLS), and negative-stain Transmission Electron Micro-
scopy (TEM) (Supplementary Table 1 and Supplementary Fig. 1).
In a rst immunization experiment, CD-1 mice (10 per group)
received two doses, three weeks apart, of the PSII-NP/VLP or PSII-
CRM conjugates (0.5 µg of PSII/dose), adjuvanted with aluminum
hydroxide (Alum). Sera were collected three weeks after the rst
dose and two weeks after the second dose for measuring IgG
titers by Luminex, using randomly biotinylated PSII as the coupling
reagent. In addition, pooled sera were evaluated in an Opsono-
phagocytic Killing Assay (OPKA) that mimics the in vivo killing of
GBS by effector cells in the presence of complement. In previous
studies, increasing OPKA titers strongly correlated with protection
after direct immunization in a neonatal mouse challenge model
and after passive transfer of maternally immunized human cord
serum to newborn mice
In all mice, preimmune IgG and OPKA titers were below the
limits of quantication (<20 for Luminex assay and <30 for OPK
assay). As shown in Table 1, after two immunizations (post-2), all
the conjugates elicited strong anti-PSII IgG and OPKA responses,
demonstrating that the tested nanoparticles were good carriers
for the capsular polysaccharide. However, PSII-Qβelicited IgG and
OPKA titers greater than the other NP- or the CRM-conjugates.
Intriguingly, a single dose of the PSII-Qβconjugate elicited IgG
and OPKA titers comparable to those elicited by two doses of PSII-
CRM or the other PSII-NP conjugates, while after one immuniza-
tion (post-1) responses in the other groups were null or very low.
A single dose of PSII-Qβelicits persistent IgG and OPKA
A second immunization experiment was conducted to conrm the
results obtained with PSII-Qβand to assess the persistence of the
immune responses after a single dose. Mice were immunized
twice with PSII-CRM on days 1 and 21, or once with PSII-Qβ, and
sera were collected at days 21, 42, 64, 99, and 134 post-
immunization for analysis of anti-PSII IgG (Luminex, individual
mouse sera) and OPKA titers (pooled sera).
As shown in Fig. 1, two doses of PSII-CRM and one dose of PSII-
Qβinduced IgG and OPKA titers that persisted until the end of the
experiment on day 134. By day 21, a single dose of PSII-Qβalready
elicited remarkably high IgG titers, and they increased along with
opsonophagocytic titers to peak on day 42 and persist out to day
134. Of note, responses were more homogeneous among animals
receiving the PSII-Qβconjugate compared to those immunized
with CRM conjugate. A further separate experiment revealed that
the responses to a single dose of PSII-CRM did not increase after
42 days but remained as low as on day 21, requiring a booster
dose to elicit functional activity in most animals (Supplementary
Fig. 2).
An additional GBS PS type conjugated to QβVLP induces
robust IgG and OPKA responses after a single dose
To assess whether a different GBS saccharide antigen conjugated
to QβVLP might also elicit a strong immune response after a
single dose, we used the same chemistry to conjugate capsular
polysaccharide Ia (PSIa) to Qβor CRM as a reference (Supple-
mentary Table 1). Mice received a single shot of PSIa-Qβor two
shots of PSIa-CRM (0.5 µg/mouse adjuvanted with Alum), and their
anti-PSIa responses were compared. While PSIa-CRM did not show
any post-1 titer, the antibody titers after one dose of the PSIa-Qβ
conjugate or two doses of PSIa-CRM conjugate were not
statistically different, and OPK titers of pooled sera were also
comparable, as shown in Fig. 2. Thus, the QβVLP proved an
optimal carrier for a single-dose GBS vaccine with two different PS
Plasmid genetic manipulation allowed to obtain a Qβcarrier
nanoparticle with highly homogenous entrapped-RNA
We tested post-2 immune responses to PSII-Qβwith and without
Alum and no signicant difference was found (Supplementary Fig.
3). This observation was not unexpected, since bacterial host RNA
randomly trapped in QβVLPs
during assembly can activate
endosomal TLR7 and 8, stimulating innate immunity that
contributes to the immunogenicity of QβVLP vaccines
. Indeed,
the Qβinfectious phage particle packages its single-stranded RNA
genome by virtue of a high-afnity interaction between a hairpin
structure and interior-facing residues of the Qβcoat protein
but can also entrap RNA derived from its host cell
. Therefore, a
tailored analytical characterization was deemed essential to
ensure consistency between different vaccine batches and their
induced immune responses.
RNA samples extracted from multiple Qβbatches were analyzed
in terms of total amount and size distribution. RNA content was
comparable between the different Qβpreparations, ranging from
20 to 30% of the total VLP mass, while microuidic capillary
electrophoresis showed non-homogenous proles with large
variations in the relative amount of RNA of different lengths
(Supplementary Fig. 4a).
Table 1. Anti-PSII IgG and OPKA titers in pooled serum samples from
mice after one and two doses of the indicated PSII conjugates
formulated with Alum where IgG titers are reported as relative
Luminex units per mL (RLU/mL) and OPKA titers as serum dilutions
mediating 50% bacterial killing.
Conjugate Post-1 Post-2
OPKA titer Anti PSII IgG
OPKA titer
PSII-CRM 587 <30 8774 432
PSII-ferritin 1295 53 8341 610
PSII-mI3 542 <30 4088 192
PSII-Qβ4160 746 15,721 2932
F. Carboni et al.
npj Vaccines (2023) 152 Published in partnership with the Sealy Institute for Vaccine Sciences
To obtain nanoparticles containing more homogeneous RNA,
we modied the E. coli expression plasmid by inserting a hairpin
structure derived from the Qβgenome immediately downstream
of the stop codon of the gene encoding the Qβcoat protein
. The
devised genetic strategy is described in Fig. 3. We named the
resulting VLPs, Qβhp
, and characterized their entrapped RNA
versus the RNA in the wild-type Qβ. The two VLPs contained
similar quantities of RNA, but the size distribution of the RNA in
the Qβhp was much more homogeneous in two independent
batches, with a major peak corresponding to the Qβcoat protein
mRNA (ca. 800 nt, Supplementary Fig. 4b).
Next, we analyzed the Qβhp and QβVLPs for the sequences of
their entrapped RNA. As shown in Fig. 4,Qβtrapped RNA was
mostly from E. coli, with a majority being rRNA and ribosomal
protein transcripts. Conversely, the RNA from Qβhp VLPs mapped
mostly to the expression plasmid, with ~55% encoding Qβcoat
protein (that further increased when the bacteria were grown in
chemically dened media), ~25% encoding other plasmid genes,
and <20% mapping to the E. coli genome. Aside from the RNA
quality, Qβand Qβhp VLPs showed no difference in purity, size, or
nal yield (>90% purity by SEC-HPLC, 16 nm radius by DLS,
23 mg/g of biomass for both).
Entrapped RNA and multivalency both contribute to
immunogenicity of PSII-Qβconjugates
To assess the carrier properties of the new construct, Qβhp VLPs
were conjugated to PSII using the same conjugation procedure as
above and analyzed by TEM and by size-exclusion HPLC, showing
comparable physico-chemical features to PSII-Qβ(Fig. 5). Groups
of 10 mice received a single dose of PSII-Qβhp or PSII-Qβ, or two
doses of PSII-CRM as reference. As shown in Fig. 6, there was no
statistical difference in IgG and OPKA titers elicited by one dose of
PSII-Qβhp or PSII-Qβ, or two doses of PSII-CRM, and antibodies
elicited by both Qβand Qβhp conjugates further increased over
In the same experiment, we investigated the adjuvant effect of
the Qβhp trapped RNA by immunizing a further group of animals
with a PSII-Qβhp conjugate containing <1% of RNA after RNAse
treatment. In mice receiving RNAse-treated PSII-Qβhp, the anti-PSII
IgG and OPKA titers on day 21 and 42 were more than one log
lower than those elicited by the untreated PSII-Qβhp, conrming
the adjuvant role of encapsidated RNA.
After the single dose of RNAse-treated PSII-Qβhp, the immune
responses increased from day 21 to 42, reaching levels similar to
those elicited by two doses of PSII-CRM (although still lower than
PSII-Qβhp). This result was presumably due to the multivalent
display of PS by the VLP, because a single dose of the classical PSII-
CRM conjugate did not cause the immune response to increase
over time (Supplementary Fig. 2).
Fig. 2 Antibody responses in mice receiving GBS PSIa-CRM or -Qβ
conjugates. PSIa IgG titers in serum samples collected from mice (20
per group) receiving two doses of PSIa-CRM or one dose of PSIa-Qβ.
The geometric mean titer (RLU/mL) is indicated by the bars,
individual mice are indicated by the dots, and the 95% condence
interval is indicated by the whiskers. IgG titers between the two
groups were compared by MannWhitney test. OPKA titers from
pooled serum samples samples of each immunization group are
reported below the barchart.
Fig. 1 Persitence of anti-PSII IgG titers in animals vaccinated with two doses of PSII-CRM and one dose of PSII-Qβconjugates. Serum
samples were collected from groups of 10 immunized mice at times after one dose of PSII-Qβor after one (day 21) or two doses of PSII-CRM
conjugates. The geometric mean titer (RLU/mL) is indicated by the bars, individual mice are indicated by the dots, and the 95% Condence
Interval is indicated by the whiskers. For GMT, non responder sera were assigned titers half of the LLOQ. The Wilcoxon signed-ranks test was
used to compare titers of the same immunization group and the MannWhitney test to compare different immunization groups. **P< 0.01;
****P< 0.0001. OPKA titers from pooled serum samples or each immunization group are reported below the histograms.
F. Carboni et al.
Published in partnership with the Sealy Institute for Vaccine Sciences npj Vaccines (2023) 152
A single dose of PSII-Qβhp VLPs elicits anti-PSII IgG and OPKA
titers greater than those elicited by a single dose of PSII-CRM
formulated with different adjuvants
Next, we assessed the possibility that vaccination with PSII-CRM
formulated with adjuvants targeting diverse Toll-like Receptors
(TLR) and immune pathways might elicit, after a single-dose, anti-
PSII IgG and OPKA titers as high or higher than those elicited by a
single dose of PSII-Qβhp. We injected mice with a single dose of
PSII-Qβhp formulated with Alum, or PSII-CRM formulated with
AS04, Poly I:C, AS37, or class B CpG, which are agonists of TLR4,
TLR3, TLR7, and TLR9, respectively. As shown in Fig. 7, the adjuvant
with PSII-CRM that elicited the highest anti-PSII IgG and OPKA
titers was AS37, a TLR7 agonist. However, titers were not as high
as those elicited by PSII-Qβhp. Thus, the adjuvancy provided by
entrapped RNA within the QβVLP was superior to that of the
adjuvants that were simply co-administered.
Self-assembling NPs are offering new avenues as vaccine delivery
vehicles for improving immune responses to subunit vaccines. As
large oligomeric structures, NPs can be engineered to display
multiple antigens in a single particle, facilitating their uptake by
antigen-presenting cells for efcient recognition and presentation
to specic surface receptors of immune cells
. Several
vaccines based on NPs and VLPs have been developed for delivery
of viral peptide and protein antigens from u
and SARS CoV-2
. Surface decoration of nanoparticles with
protein antigens has also shown promise for application to
bacterial vaccines
. Recent studies have investigated NPs and
VLPs as carriers of bacterial saccharide antigens with the aim of
improving T-cell help to anti-glycan B cells. Polonskaya and
colleagues pioneered the display of S. pneumoniae synthetic tetra-
saccharides of serotypes 3 and 14 on the surface of VLPs, resulting
in strong and persistent post-2 and post-3 serotype-specic IgG
responses that protected mice against infectious challenge
More recently, a modular biosynthetic approach to produce
nanoconjugate vaccines using the SpyTag/SpyCatcher system was
applied for the direct coupling of Shigella exneri native
polysaccharides to AP205 and QβVLPs. The authors demonstrated
an increase in Tfh and germinal center B cells in draining lymph
nodes, compared to classical protein carrier conjugates, and an
efcient adaptive immune response and prophylaxis against
bacterial challenge
We investigated the possibility to develop a GBS vaccine for a
single administration during the third trimester of pregnancy,
leveraging NPs capability to elicit potent immune responses.
Capsular polysaccharide II was chemically conjugated to Ferritin
and mI3 NPs and QβVLPs. After vaccinating mice with two doses,
all of these nanoconjugates elicited PSII-specic antibody func-
tional responses higher than the reference CRM conjugate,
conrming that self-assembling NPs are potent delivery systems
for polysaccharide antigens that can be exploited as an alternative
to classical and widely used carrier proteins
. The Qβconjugate
elicited post-1 responses higher than the other nanoconjugates
and like those obtained after two doses of PSII-CRM, conrming
QβVLP as a suitable system to enhance responses against poorly
immunogenic saccharide targets. Further, IgG and OPKA titers
induced by the PSII-Qβconjugate reached a maximum on day 42
and persisted up to day 134. The immune-enhancement of the Qβ
carrier was further conrmed with capsular polysaccharide Ia
(PSIa), suggesting the same approach could be applicable and
tested for the other CPS.
Our data are also in line with previous literature showing that
Qβ-VLPs can enhance immune responses to other weak or non-
immunogenic antigens like small peptides and glycopeptides, as
demonstrated for human vaccines against nicotine dependence,
hypertension, allergies, diabetes, cancer, and Alzheimers disease
and are safe and highly immunogenic
. Further, this study
conrm the self-adjuvant effect of VLPs associated with the
presence of entrapped RNA, which acts as a scaffold for the self-
assembly of prophage coat protein monomers, and activates the
innate immune system via TLR7 and 8
Fig. 3 Genetic strategy used for the generation of Qβand Qβhp VLPs. Top panel: expression of QβVLPs was achieved by cloning the Qβ
coat protein open-reading frame under the T7 promoter; monomers self-assemble drandomly incorporating E.coli RNA. Bottom panel: for
Qβhp VLPs expression, a hairpin structure was inserted immediately downstream of the stop codon of the gene encoding the Qβcoat protein;
monomers self-assembled incorporating mainly Qβhp RNA.
F. Carboni et al.
npj Vaccines (2023) 152 Published in partnership with the Sealy Institute for Vaccine Sciences
Encapsulated RNA represents for about 2030% of the QβVLP
mass and mainly consists of hairpin-rich molecules derived from
the prophage or encoded in the bacterial host genome
resulting in fragments of highly variable size and composition.
Rhee and colleagues demonstrated that RNA encoding the green
uorescent protein (GFP) could be delivered into QβVLPs by
adding a hairpin sequence to the end of its coding gene
achieve a more reproducible RNA prole of our VLPs, we designed
an E. coli strain where the hairpin sequence was added to the
plasmid-encoded Qβcoat protein gene, resulting in a VLP
containing RNA with a more homogeneous prole, mainly
represented by a single major peak encoding the Qβcoat protein,
the new Qβhp
Like its precursor, PSII-Qβ, the PSII-Qβhp conjugate induced
high IgG and OPKA titers after a single vaccine dose. To conrm
the effect of the VLP-entrapped RNA as immune potentiator and
to better understand if the VLP size and ability to present multiple
copies of the target antigen on its surface could also play a role in
the observed enhanced immune responses, RNA was almost
completely removed from the PSII-Qβhp conjugate by RNAse
treatment. This caused a strong decrease in anti-PSII immune
responses; however, 42 days after a single dose of the RNA-
depleted VLP conjugate, antibody levels were like those in animals
receiving two doses of PSII-CRM. We concluded that the
entrapped RNA and multivalent antigen presentation of the Qβ
VLP both contribute to achieve optimal immune response. To
support this hypothesis, we compared PSII-Qβhp with the PSII-
CRM conjugate formulated with AS04, Poly I:C, AS37, or class B
CpG (agonists of TLR4, TLR3, TLR7, and TLR 9, respectively)
single dose of PSII-CRM co-administered with these adjuvants did
not induce IgG and OPKA responses comparable to those
observed with PSII-Qβ, suggesting that the presentation of the
antigen and the immune potentiator in a single VLP offers an
advantage compared to administering them as separate compo-
nents in the same formulation.
The immune-enhancing effect of Qβas antigen carrier has been
appreciated beyond rodent species. Phares and colleagues
compared the immunogenicity of malaria candidate vaccines
containing a recombinant circumsporozoite protein or derived
polypeptides, either in soluble form or conjugated to Qβ, both in
mice and in non-human primates. Interestingly, they found higher
antibody titers after a single dose of the VLP conjugates than after
a single dose of the unconjugated antigens in both species
In conclusion, QβVLPs offer promise as carriers for the delivery
of saccharide antigens against group B Streptococcus and beyond,
and can be particularly attractive for the development of single-
dose vaccines.
Expression and purication of nanoparticles
The genes encoding for ferritin, mI3 and Qβwere synthesized as
DNA strings by GeneArt (Thermo Fisher Scientic) optimizing the
codon usage for expression in the E. coli. The genes were cloned
into pET15b+TEV and pET21b+(Merck-Sigma) PCR-amplied
vectors using the Infusion cloning kit (Takara) following manu-
facturer instructions
. The plasmid pET24b+encoding the
Qβhp coat protein was purchased by GeneArt (Thermo Fisher
All recombinant proteins were expressed in E. coli BL21(DE3)
(New England Biolabs). For ml3 expression, cells were grown in
HTMC medium (Glycerol 15 g/L; Yeast Extract 30 g/L; MgSO
O 0.5 g/L; KH
5 g/L; K
20 g/L; KOH 1 M to pH nal
7.35 ± 0.1) supplemented with Kanamycin, under shaking at 20 °C
for 16 h followed by induction with 1 mM IPTG for 24 h. Qβand
Qβhp particles were produced using a 5 L bioreactor (Applikon)
set at controlled temperature of 32 °C, pH 6.50 ± 0.1 (by the
addition of 4 M NaOH and 2 M H
when needed), 400800
RPM, air ow rate between 12 VVM and 30%100% dissolved
oxygen (by automatic enrichment of the inlet air stream with Air
and RPM). For the production of Qβand Qβhp lot 1 host E. coli
cells were grown in HTMC; when an OD
value of 5 was reached,
Induction Solution (Glycerol 30 g/l; CaCl
0.12 g/L; MgSO
0.15 g/L;
IPTG 1 mM) was added to the culture and the biomass recovered
after 5 h. For Qβhp lot 2 production, bacteria were cultured in M9
modied medium (Glycerol 30 g/L; NH
Cl 3 g/L; KH
14 g/L;
6 g/L; MgSO
0.24 g/L; Riboavin 0.002 g/L; Thiamine
0.002 g/L; Pyridoxine hydrochloride 0.002 g/L; Nicotinamide
0.002 g/L; FeSO
1.53 mg/L; MnCl
2.64 mg/L; CuSO
1.98 mg/L;
0.34 mg/L; Na
0.6 mg/L); when an OD
value of 3
was reached, induction solution (Glycerol 30 g/l; CaCl
0.12 g/L;
0.15 g/L; (NH
6 g/L; FeSO
9.18 mg/L; MnCl
15.84 mg/L; CuSO
6.78 mg/L; ZnSO
11.88 mg/L; Na
2.04 mg/L; IPTG 1 mM) was added and the biomass recovered.
For mI3 purication, bacterial cells were chemically lysed with
CelLytic Express (SIGMA), followed by several steps of purication,
in sequence: Afnity Chromatography Hi Trap HP, Ni
Size Exclusion Chromatography Sephacryl S300 (Cytiva), Endotrap
Column RED (IlexLife) and Hydroxyapatite column CHT (BIO-RAD)
for endotoxin removal. Pooled fractions were stored in 100 mM
Fig. 4 Sequencing analysis of RNA extracted from Qβand Qβhp.
Quantication of the different categories of RNA-seq reads is
indicated as a percentage of the total number of reads in samples
from 1 lot of Qβ(rst lane) and 2 lots of Qβhp (second and third
lanes). In the bar plot, shades of blue are used for reads mapping to
the E. coli BL21DE3 genomic sequence and shades of yellow and
brown for reads mapping to the pET24b+plasmid. lacI is reported
in green as it is present in two copies on the genome and in one
copy on the plasmid.
F. Carboni et al.
Published in partnership with the Sealy Institute for Vaccine Sciences npj Vaccines (2023) 152
pH7,6 at 20 °C until further use. For Qβand Qβhp
purication, bacterial cells were subjected to mechanical lysis, and
the supernatant occulated with Polyethyleneimine (PEI). After
centrifugation, the occulate containing Qβor Qβhp was puried
by Ionic Exchange Chromatography (Cytiva), followed by Size
Exclusion Chromatography Sephacryl S500 (Cytiva) in PBS; the
puried VLPs were stored at 20 °C until further use. Protein
concentration was determined by PierceRapid Gold BCA Protein
Assay Kit (Thermo Fisher Scientic, Illinois, USA) according to
manufacturers instructions, using Bovine Serum Albumin (BSA) as
Analytical characterization of Qβand Qβhp VLPs
Size exclusion chromatography was performed on a TSKgel
G6000PW column (17 µm, 7.5 × 300 mm, Tosoh, Japan) using an
Alliance Bio HPLC system (Waters, Massachussets, USA) equipped
with a photodiode array detector and a multiple-wavelength
uorescence detector. Analysis was performed with an isocratic
mobile phase of PBS, at 0.5 mL/min, at room temperature. A multi-
angle light scattering (MALS) system was coupled downstream of
the HPLC. MALS signals were detected by a Dawn Heleos II
detector and an Optilab T-rEX refractive index (RI) detector (Wyatt,
0 4 8 12 16 20 24 28 32 36
SE-HPLC analysis
0 4 8 12 16 20 24 28 32 36
SE-HPLC analysis
Fig. 5 Analytical characterization of PSII-Qβ(left) and PSII-Qβhp conjugates (right). a Transmission electron microscopy (TEM) in negative
staining, and bSize-exclusion High Performance Liquid Chromatography (SE-HPLC).
Fig. 6 Immune responses in mice receiving PSII conjugated to different QβVLPs. Mice (10 per group) received one (full bars) or two doses
(patterned bar) of PSII-CRM, PSII-Qβ, PSII-Qβhp, or RNAse-treated PSII-Qβhp. The geometric mean IgG titer (RLU/mL) is indicated by the bars,
individual mice are indicated by the dots, and the 95% Condence Interval is indicated by the whiskers. For GMT, non responder sera were
assigned titers half of the LLOQ. The Wilcoxon signed-ranks test was used to compare titers at day 21 and day 42 of the same immunization
group of the same immunization group and the KruskalWallis and Dunn multiple comparisons test was used to compare different
immunization groups. **P< 0.01. The corresponding OPKA titers from pools of serum samples are reported below the barchart.
F. Carboni et al.
npj Vaccines (2023) 152 Published in partnership with the Sealy Institute for Vaccine Sciences
Santa Barbara, CA). ASTRA 7.3.1 software was used for acquiring
and analyzing UV, RI, and MALS data.
VLP Particle Size Distribution was assessed by DLS with the
DynaPro PlateReader-II (Wyatt Technology, California, USA) using
80 µL of sample/well and 35 measurements/sample.
For Transmission Electron Microscopy analysis, 5 µL of samples
(20 ng/µL) in PBS were loaded for 30 s on a glow discharger
copper 200-square mesh grids (EMS). Blotted the excess, the grid
was negatively stained using NanoW (Nanoprobes) for 30 s and let
air dried. Samples were analyzed by a Tecnai G2 spirit and images
acquired using a Tvips TemCam-F216 (EM-Menu software).
Analysis of Qβand Qβhp RNA content
One hundred µg of puried VLPs were lysed with 600 µL of Trizol
at RT for 5 min and 600 µL of absolute ethanol were added to the
solution. RNA was extracted by Pure link RNA Mini Kit according to
the manufacturers instructions. Total RNA was quantied by
Quant-iTRiboGreen®RNA Reagent and Kit (Molecular Probes®,
Oregon, USA), using rRNA as standard. Native (untreated) and
denaturated (by incubation with 4 M urea for 30 min at 95 °C) Qβ
VLPs were diluted in TE buffer (10 mM Tris-HCl, EDTA, pH 7.5) and
100 µL of RiboGreen were added to 100 µL of sample; uores-
cence intensity (Excitation/Emission 485 nm/530 nm) was mea-
sured (Innite 200, Tecan). RNA total content was expressed as µg/
mL or % RNA/Protein.
For capillary electrophoresis analysis, 20 mL of puried RNA
samples were loaded onto LabCHip GX II (PerkinElmer) following
manufacturers instructions. Each sample was analyzed in
For sequencing analysis, three RNA libraries were prepared from
each sample starting with 100 ng of RNA (quantied by Agilent
Bioanalyzer RNA 6000 Nano Kit) following the Illumina protocol
Stranded Total RNA Prep with Ribo-Zero Plus (Illumina, San Diego,
CA). The ribosomal RNA depletion step was omitted. Samples were
sequenced on Miseq Illumina platform PE 2 × 75 cycles. Reads
quality was checked with fastQC version 0.11.9. As reference for
mapping we used E. coli BL21(DE3) reference genome down-
loaded from the NCBI (GenBank accession: GCA_013167015.1)
coupled with genetic sequences of pET 19ABXGYC and
20ADXZPC, downloaded from the vendor website. Reads were
aligned with bowtie2 version 2.4.1 and counted using samtools
version 1.9 and bedtools version 2.29.2.
Conjugation of GBS polysaccharides to nanoparticles
GBS polysaccharide II or Ia were oxidized targeting 20% of sialic
acid residues
. Samples were stirred with 0.1 M sodium periodate
in 10 mM sodium phosphate buffer in the dark, for 2 h at room
temperature. The mixture was puried by liquid chromatography
using G-25 desalting columns. The oxidized polysaccharide was
dissolved in a 100 mM sodium phosphate buffer at pH 7.2 and
mixed to NPs at a nal protein concentration of about 10 mg/mL.
Sodium cyanoborohydride (1:1 w/w based on the amount of
polysaccharide) was added to the solution. The reaction mixture
was incubated at 37 °C for 3 days, quenched with sodium
borohydride and puried by tangential ow ltration (Sartocon
100 kDa lter). The protein content was determined by BCA
colorimetric assay. Finally, the extent of saccharide conjugation
was evaluated by SDS-PAGE electrophoresis and SE-HPLC using a
Sepax SRT-C 2000 column.
PSII-Qβconjugates devoid of RNA were generated by incuba-
tion of the samples with an RNase Cocktail (Invitrogen), mixture of
two highly puried ribonucleases, RNase A and RNase T1. After an
overnight incubation at room temperature, RNAses and nucleic
acids were eliminated by serial centrifugal ltration (100 kDa).
Saccharide quantication in the conjugates was performed by
high-performance anion-exchange chromatography with pulsed
amperometric detection (HPAEC-PAD). GBS PSII or PSIa standard
samples at ve increasing concentrations ranging between 0.5
and 10 μg/mL, were prepared to build the calibration curve. Test
samples were diluted targeting the calibration curve midpoint,
Fig. 7 Immune responses in mice receiving PSII-CRM with different adjuvants. Mice (10 per group) received a single dose of PSII-CRM
formulated with AS04, Poly I:C, AS37 and CpG or PSII-Qβhp formulated with Aluminum hydroxide. The geometric mean IgG titer (RLU/mL) at
day 21 and 42 is indicated by the bars, individual mice are indicated by the dots, and the 95% condence interval is indicated by the whiskers.
For GMT, non responder sera were assigned titers half of the LLOQ. The KruskalWallis and Dunn multiple comparisons test was used to
compare the group receiving PSII-Qβhp with all PSII-CRM immunization groups. **P< 0.01; ****P< 0.0001. The OPKA titers from pools of serum
samples at day 21 are reported below the barchart.
F. Carboni et al.
Published in partnership with the Sealy Institute for Vaccine Sciences npj Vaccines (2023) 152
while free saccharide samples were analyzed undiluted after
separation by solid-phase extraction (SPE) cartridges. The refer-
ence and conjugate samples were prepared in 4 M triuoroacetic
acid (Supelco), incubated at 100 °C for 3 h, dried under vacuum
(SpeedVac Thermo), suspended in water, and ltered with 0.45 μm
Phenex-NY (Phenomenex) lters. HPAEC-PAD analysis was per-
formed with a Dionex ICS-6000 equipped with a CarboPac PA1
column (4 × 250 mm; Dionex) coupled with a PA1 guard to column
(4 × 50 mm; Dionex). Samples were run at 1 mL/min, using an
isocratic elution with 14 mM NaOH, followed by a washing step
with 500 mM NaOH. The efuent was monitored using an
electrochemical detector in the pulse amperometric mode, with
a gold working electrode and an Ag/AgCl reference electrode. A
quadruple-potential waveform for carbohydrates was applied. The
resulting chromatographic data were processed using Chrome-
leon software 7.2 (Thermo Dionex), and sample concentrations
were determined based on galactose content.
In vivo experiments
Animal studies have been approved by the Italian Ministry of
Health and were ethically reviewed by the local Animal Welfare
Body. Animal studies were carried out at GSK facility in accordance
with national/European legislation and the GSK Policies on the
Care, Welfare and Treatment of Animals. Groups of 10 CD1 (ICR)
68 weeks old female mice, were immunized intramuscularly (IM)
with GBS conjugates (0.5 μg of PS/dose) adjuvanted with
Aluminum Hydroxide (2 mg/mL AlumOH in Histidine buffer
pH6.5, 150 mM NaCl). In one of the experiments CRM conjugates
were alternatively formulated with AS04 2 mg/mL (MPL based
200 μg/mL), AS37 (LHD153R based 200 μg/mL), Poly I:C (30 μg/
dose) or CpG class B (30 μg/dose).
Formulates with Poly I:C (30 μg/dose) and CpG class B (30 μg/
dose) were prepared in PBS pH7.4, those with AS04 and AS37 in
Histidine buffer pH6.5, 150 mM NaCl. All formulations were
controlled for pH, osmolality, presence of precipitates, antigen
adsorption, endotoxin content and bioburden.
Biotin-PS II immunoassay
The immune response to GBS PS conjugates was assessed by
Luminex-based monoplex assay
using Biotin-PS II or Ia (1 µg/mL)
conjugated to 1.25 × 106 Radix High capacity (HC) Streptavidin
magnetic beads. Eight steps of threefold dilutions of a standard
serum (pool of sera from mice immunized with PSII-CRM used as
reference) and samples were mixed with an equal volume of
conjugated Biotin-PS beads (3000 beads/well) in a 96-well Greiner
(Millipore Corporation, Billerica, MA) and incubated for 60 min at
RT in the dark on a plate shaker at 600 rpm. After incubation, the
beads were washed three times with 200 µl PBS. Each well was
then loaded with 50 µl of a 1/100 dilution of PE-secondary anti-
mouse IgG (Jackson Immunoresearch, 115-116-072) and the plates
were incubated for 60 min with continued shaking. After washing,
beads were suspended in 100 µL of PBS before the analysis with
Bioplex 200. Data were acquired in real time by Bioplex Manager
Software 6.2 (Bio-Rad Laboratories, Hercules, CA).
The median uorescence intensity (MFI) was converted to RLU/
mL by interpolation to the corresponding 5-PL Standard curve (8
dilution points). IgG concentrations (RLU/mL) were determined
from the mean of two sample dilutions for which MFI signals were
in the linear range of the standard curve.
Opsonophagocytic killing assay (OPKA)
OPKA was conducted using differentiated HL-60 cells and the GBS
strain DK21 (serotype II)
. OPK titers were expressed as the
reciprocal serum dilution mediating 50% bacterial killing, esti-
mated through piecewise linear interpolation of the dilution-
killing OPK data. A uorescent OPKA was adapted from
to increase the throughput capacity to 384-wells
and to avoid CFU counting by introducing a uorescent dye
(Alamar Blue) that allow to measure the viability endpoint of
bacteria through a plate reader. The uorescent OPKA used the
same assay components and titration method. The lower limit of
detection was 1:30 dilution and the assay coefcient of variation
was ~30% for both assay formats.
The authors declare that data supporting the ndings of this study are available
within the paper and its supplementary information les. All relevant data are
available from the authors.
Received: 23 June 2023; Accepted: 15 September 2023;
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The authors thank Ilaria Ferlenghi, Federico Fontani, Alessandra Anemona, Evita
Balducci, and Marta Tontini for contributing to antigen preparation and characteriza-
tion, and for the design of mouse studies and statistics.
Conceived and designed the experiments: F.C., D.M., B.W., I.M., and M.R.R. Performed
the experiments: F.C., R.C., G.R., G.T., C.B., G.B., M.G., C.B., F.G., S.M., G.B., B.B., P.C., L.C.,
C.N., S.S., and C.F. Analyzed the data: F.C., R.C., G.R., G.T., C.B., G.B., M.G., C.B., F.G., S.M.,
G.B., B.B., E.F., M.F., D.S., S.S., D.M., B.W., I.M., and M.R.R. Contributed to the writing of
the manuscript: F.C., I.M., and M.R.R. All authors have read, revised, and agreed to the
published version of the manuscript.
This work was undertaken at the request of and sponsored by GlaxoSmithKline. F.C.,
R.C., G.R., G.T., C.B., G.B., M.G., C.B., F.G., S.M., G.B., B.B., P.C., L.C., C.N., S.S., C.F., E.F., M.F.,
D.S., S.S., D.M., B.W., I.M., and M.R.R. are employees of the GSK group of companies.
F. Carboni et al.
Published in partnership with the Sealy Institute for Vaccine Sciences npj Vaccines (2023) 152
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available at
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Full-text available
The presentation of viral antigens on nanoparticles in multivalent arrays has emerged as a valuable technology for vaccines. On the nanoparticle surface, highly ordered, repetitive arrays of antigens can mimic their geometric arrangement on virion surfaces and elicit stronger humoral responses than soluble viral antigens. More recently, bacterial antigens have been presented on self-assembling protein nanoparticles and have elicited protective antibody and effective T-helper responses, further supporting the nanoparticle platform as a universal approach for stimulating potent immunogenicity. Here, we present the rational design, structural analysis, and immunogenicity of self-assembling ferritin nanoparticles displaying eight copies of the Neisseria meningitidis trimeric adhesin NadA. We engineered constructs consisting of two different NadA fragments, head only and head with stalk, that we fused to ferritin and expressed in Escherichia coli. Both fusion constructs self-assembled into the expected nanoparticles as determined by Cryo electron microscopy. In mice, the two nanoparticles elicited comparable NadA antibody levels that were 10- to 100-fold higher than those elicited by the corresponding NadA trimer subunits. Further, the NadAferritin nanoparticles potently induced complement-mediated serum bactericidal activity. These findings confirm the value of self-assembling nanoparticles for optimizing the immunogenicity of bacterial antigens and support the broad applicability of the approach to vaccine programs, especially for the presentation of trimeric antigens.
Full-text available
Group B streptococcus (GBS) is a leading cause of life-threatening neonatal infections and subsets of adverse pregnancy outcomes. Essentially all GBS strains possess one allele of the alpha-like protein (Alp) family. A maternal GBS vaccine, consisting of the fused N-terminal domains of the Alps αC and Rib (GBS-NN), was recently demonstrated to be safe and immunogenic in healthy adult women. To enhance antibody responses to all clinically relevant Alps, a second-generation vaccine has been developed (AlpN), also containing the N-terminal domain of Alp1 and the one shared by Alp2 and Alp3. In this study, the safety and immunogenicity of AlpN is assessed in a randomized, double-blind, placebo-controlled, and parallel-group phase I study, involving 60 healthy non-pregnant women. AlpN is well tolerated and elicits similarly robust and persistent antibody responses against all four Alp-N-terminal domains, resulting in enhanced opsonophagocytic killing of all Alp serotypes covered by the vaccine.
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Recombinant protein-based vaccines are a valid and safer alternative to traditional vaccines based on live-attenuated or killed pathogens. However, the immune response of subunit vaccines is generally lower compared to that elicited by traditional vaccines and usually requires the use of adjuvants. The use of self-assembling protein nanoparticles, as a platform for vaccine antigen presentation, is emerging as a promising approach to enhance the production of protective and functional antibodies. In this work we demonstrated the successful repetitive antigen display of the C-terminal β-barrel domain of factor H binding protein, derived from serogroup B Meningococcus on the surface of different self-assembling nanoparticles using genetic fusion. Six nanoparticle scaffolds were tested, including virus-like particles with different sizes, geometries, and physicochemical properties. Combining computational and structure-based rational design we were able generate antigen-fused scaffolds that closely aligned with three-dimensional structure predictions. The chimeric nanoparticles were produced as recombinant proteins in Escherichia coli and evaluated for solubility, stability, self-assembly, and antigen accessibility using a variety of biophysical methods. Several scaffolds were identified as being suitable for genetic fusion with the β-barrel from fHbp, including ferritin, a de novo designed aldolase from Thermotoga maritima , encapsulin, CP3 phage coat protein, and the Hepatitis B core antigen. In conclusion, a systematic screening of self-assembling nanoparticles has been applied for the repetitive surface display of a vaccine antigen. This work demonstrates the capacity of rational structure-based design to develop new chimeric nanoparticles and describes a strategy that can be utilized to discover new nanoparticle-based approaches in the search for vaccines against bacterial pathogens.
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The coat proteins (CPs) of single-stranded RNA bacteriophages (ssRNA phages) directly assemble around the genomic RNA (gRNA) to form a near-icosahedral capsid with a single maturation protein (Mat) that binds the gRNA and interacts with the retractile pilus during infection of the host. Understanding the assembly of ssRNA phages is essential for their use in biotechnology, such as RNA protection and delivery. Here, we present the complete gRNA model of the ssRNA phage Qβ, revealing that the 3′ untranslated region binds to the Mat and the 4127 nucleotides fold domain-by-domain, and is connected through long-range RNA–RNA interactions, such as kissing loops. Thirty-three operator-like RNA stem-loops are located and primarily interact with the asymmetric A/B CP-dimers, suggesting a pathway for the assembly of the virions. Additionally, we have discovered various forms of the virus-like particles (VLPs), including the canonical T = 3 icosahedral, larger T = 4 icosahedral, prolate, oblate forms, and a small prolate form elongated along the 3-fold axis. These particles are all produced during a normal infection, as well as when overexpressing the CPs. When overexpressing the shorter RNA fragments encoding only the CPs, we observed an increased percentage of the smaller VLPs, which may be sufficient to encapsidate a shorter RNA.
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Virus-like particles (VLPs) are nanostructures that possess diverse applications in therapeutics, immunization, and diagnostics. With the recent advancements in biomedical engineering technologies, commercially available VLP-based vaccines are being extensively used to combat infectious diseases, whereas many more are in different stages of development in clinical studies. Because of their desired characteristics in terms of efficacy, safety, and diversity, VLP-based approaches might become more recurrent in the years to come. However, some production and fabrication challenges must be addressed before VLP-based approaches can be widely used in therapeutics. This review offers insight into the recent VLP-based vaccines development, with an emphasis on their characteristics, expression systems, and potential applicability as ideal candidates to combat emerging virulent pathogens. Finally, the potential of VLP-based vaccine as viable and efficient immunizing agents to induce immunity against virulent infectious agents, including, SARS-CoV-2 and protein nanoparticle-based vaccines has been elaborated. Thus, VLP vaccines may serve as an effective alternative to conventional vaccine strategies in combating emerging infectious diseases.
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In the last decade, the interest in ferritin-based vaccines has been increasing due to their safety and immunogenicity. Candidates against a wide range of pathogens are now on Phase I clinical trials namely for influenza, Epstein-Barr, and SARS-CoV-2 viruses. Manufacturing challenges related to particle heterogeneity, improper folding of fused antigens, and antigen interference with intersubunit interactions still need to be overcome. In addition, protocols need to be standardized so that the production bioprocess becomes reproducible, allowing ferritin-based therapeutics to become readily available. In this review, the building blocks that enable the formulation of ferritin-based vaccines at an experimental stage, including design, production, and purification are presented. Novel bioengineering strategies of functionalizing ferritin nanoparticles based on modular assembly, allowing the challenges associated with genetic fusion to be circumvented, are discussed. Distinct up/down-stream approaches to produce ferritin-based vaccines and their impact on production yield and vaccine efficacy are compared. Finally, ferritin nanoparticles currently used in vaccine development and clinical trials are summarized.
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The rapid development of a SARS-CoV-2 vaccine is a global priority. Here, we develop two capsid-like particle (CLP)-based vaccines displaying the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. RBD antigens are displayed on AP205 CLPs through a split-protein Tag/Catcher, ensuring unidirectional and high-density display of RBD. Both soluble recombinant RBD and RBD displayed on CLPs bind the ACE2 receptor with nanomolar affinity. Mice are vaccinated with soluble RBD or CLP-displayed RBD, formulated in Squalene-Water-Emulsion. The RBD-CLP vaccines induce higher levels of serum anti-spike antibodies than the soluble RBD vaccines. Remarkably, one injection with our lead RBD-CLP vaccine in mice elicits virus neutralization antibody titers comparable to those found in patients that had recovered from COVID-19. Following booster vaccinations, the virus neutralization titers exceed those measured after natural infection, at serum dilutions above 1:10,000. Thus, the RBD-CLP vaccine is a highly promising candidate for preventing COVID-19. Here the authors generate a capsid-like particle based vaccine candidate displaying the receptor-binding domain of the SARS-CoV-2 spike protein and show induction of neutralizing antibodies after intramuscular prime-boost immunization in mice.
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Antigens displayed on self-assembling nanoparticles can stimulate strong immune responses and have been playing an increasingly prominent role in structure-based vaccines. However, the development of such immunogens is often complicated by inefficiencies in their production. To alleviate this issue, we developed a plug-and-play platform using the spontaneous isopeptide-bond formation of the SpyTag:SpyCatcher system to display trimeric antigens on self-assembling nanoparticles, including the 60-subunit Aquifex aeolicus lumazine synthase (LuS) and the 24-subunit Helicobacter pylori ferritin. LuS and ferritin coupled to SpyTag expressed well in a mammalian expression system when an N-linked glycan was added to the nanoparticle surface. The respiratory syncytial virus fusion (F) glycoprotein trimer—stabilized in the prefusion conformation and fused with SpyCatcher—could be efficiently conjugated to LuS-SpyTag or ferritin-SpyTag, enabling multivalent display of F trimers with prefusion antigenicity. Similarly, F-glycoprotein trimers from human parainfluenza virus-type 3 and spike-glycoprotein trimers from SARS-CoV-2 could be displayed on LuS nanoparticles with decent yield and antigenicity. Notably, murine vaccination with 0.08 µg of SARS-CoV-2 spike-LuS nanoparticle elicited similar neutralizing responses as 2.0 µg of spike, which was ~ 25-fold higher on a weight-per-weight basis. The versatile platform described here thus allows for multivalent plug-and-play presentation on self-assembling nanoparticles of trimeric viral antigens, with SARS-CoV-2 spike-LuS nanoparticles inducing particularly potent neutralizing responses.
Conjugate vaccines represent one of the most effective means for controlling the occurrence of bacterial diseases. Although nanotechnology has been greatly applied in the field of vaccines, it is seldom used for conjugate vaccine research because it is very difficult to connect polysaccharides and nanocarriers. In this work, an orthogonal and modular biosynthesis method was used to produce nanoconjugate vaccines using the SpyTag/SpyCatcher system. When SpyTag/SpyCatcher system is combined with protein glycosylation technology, bacterial O-polysaccharide obtained from Shigela flexneri 2a can be conjugated onto the surfaces of different virus-like particles (VLPs) in a biocompatible and controlled manner. After confirming the excellent lymph node targeting and humoral immune activation abilities, these nanoconjugate vaccines further induced efficient prophylactic effects against infection in a mouse model. These results demonstrated that natural polysaccharide antigens can be easily connected to VLPs to prepare highly efficient nanoconjugate vaccines. To the best of the researchers' knowledge, this is the first time VLP-based nanoconjugate vaccines are produced efficiently, and this strategy could be applied to develop various pathogenic nanoconjugate vaccines. Electronic supplementary material: Supplementary material (Figs. S1-S9) is available in the online version of this article at 10.1007/s12274-021-3713-4.
Adjuvants are vaccine components that enhance the magnitude, breadth and durability of the immune response. Following its introduction in the 1920s, alum remained the only adjuvant licensed for human use for the next 70 years. Since the 1990s, a further five adjuvants have been included in licensed vaccines, but the molecular mechanisms by which these adjuvants work remain only partially understood. However, a revolution in our understanding of the activation of the innate immune system through pattern recognition receptors (PRRs) is improving the mechanistic understanding of adjuvants, and recent conceptual advances highlight the notion that tissue damage, different forms of cell death, and metabolic and nutrient sensors can all modulate the innate immune system to activate adaptive immunity. Furthermore, recent advances in the use of systems biology to probe the molecular networks driving immune response to vaccines (‘systems vaccinology’) are revealing mechanistic insights and providing a new paradigm for the vaccine discovery and development process. Here, we review the ‘known knowns’ and ‘known unknowns’ of adjuvants, discuss these emerging concepts and highlight how our expanding knowledge about innate immunity and systems vaccinology are revitalizing the science and development of novel adjuvants for use in vaccines against COVID-19 and future pandemics. This Review discusses how recent advances in understanding the activation of the innate immune system are shedding light on the immunological mechanisms of action of adjuvants and highlights how systems-based approaches are beginning to revitalize adjuvant design and development.