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

Abstract

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.

Full text

ARTICLE OPEN
Proof of concept for a single-dose Group B Streptococcus
vaccine based on capsular polysaccharide conjugated to Qβ
virus-like particles
Filippo Carboni
1
, Roberta Cozzi
1
, Giacomo Romagnoli
1
, Giovanna Tuscano
1
, Cristiana Balocchi
1
, Giada Buffi
1
, Margherita Bodini
1
,
Cecilia Brettoni
1
, Fabiola Giusti
1
, Sara Marchi
1
, Giulia Brogioni
1
, Barbara Brogioni
1
, Paolo Cinelli
1
, Luigia Cappelli
1
, Chiara Nocciolini
1
,
Silvia Senesi
1
, Claudia Facciotti
1
, Elisabetta Frigimelica
1
, Monica Fabbrini
1
, Daniela Stranges
1
, Silvana Savino
1
, Domenico Maione
1
,
Benjamin Wizel
2
, Immaculada Margarit
1
✉and Maria Rosaria Romano
1
✉
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.
npj Vaccines (2023) 8:152 ; https://doi.org/10.1038/s41541-023-00744-5
INTRODUCTION
Streptococcus agalactiae (Group B Streptococcus, GBS) has adapted
to human entero-genital environments and colonizes 15–30% of
women
1
. During pregnancy, these bacteria can reach the fetus
and cause stillbirth or preterm delivery
2
. Both pre-partum and
peripartum infections can result in severe neonatal bacteremia,
sepsis, and/or meningitis
3
. The risk of Early-Onset GBS disease in
the first week of life has been reduced by intrapartum antibiotic
prophylaxis
4
. 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 first three
months
5
, and it is difficult to implement in poor-resourced
countries
6
. 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
7,8
. Each PS when conjugated to carrier
proteins and administered to female mice prior to mating, elicited
serotype-specific functional antibodies that were efficiently
transferred to pups and mediated their protection against GBS
lethal challenge
9
. In humans, PS serotype-specific maternal
antibody titers inversely correlated with the risk for infection in
infants
10
, providing the basis to develop a PS-based maternal
vaccine for protecting neonates
11
. 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
12
.
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
studies
13–17
. 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-specific IgG responses
were very high in individuals with pre-immune titers above the
Limit of Quantification (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
18
, while a second dose offered more than 1
year apart strongly boosted PS-specific responses
13,19
. 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
20,21
and in humans
22
.
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
23
. Several vaccines based on NPs and VLPs
have been developed for delivery of viral peptide and protein
antigens
24–30
.
NPs and VLPs are also receiving growing interest as carriers of
polypeptide antigens for developing vaccines against bacterial
pathogens
31–34
. However, they have been less studied as carriers
1
GSK, Siena, Italy.
2
GSK, Rockville, MD, USA. ✉email: immaculada.x.margarit-y-ros@gsk.com; maria.r.romano@gsk.com
www.nature.com/npjvaccines
Published in partnership with the Sealy Institute for Vaccine Sciences
1234567890():,;

of saccharide antigens
35,36
. Here we compared different self-
assembling NPs and VLPs as scaffolds for GBS PS antigens to
improve their immunogenicity after a single vaccine dose.
RESULTS
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
9,37
, and non-human primates
38
. 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
39
and mI3
40
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
8
. The resulting PSII
conjugates were purified 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 confirmed 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 first 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 first
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
12
.
In all mice, preimmune IgG and OPKA titers were below the
limits of quantification (<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
responses
A second immunization experiment was conducted to confirm 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
types.
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 significant difference was found (Supplementary Fig.
3). This observation was not unexpected, since bacterial host RNA
randomly trapped in QβVLPs
41
during assembly can activate
endosomal TLR7 and 8, stimulating innate immunity that
contributes to the immunogenicity of QβVLP vaccines
42
. Indeed,
the Qβinfectious phage particle packages its single-stranded RNA
genome by virtue of a high-affinity interaction between a hairpin
structure and interior-facing residues of the Qβcoat protein
43,44
,
but can also entrap RNA derived from its host cell
41
. 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 microfluidic capillary
electrophoresis showed non-homogenous profiles 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
Anti PSII IgG
titer
OPKA titer Anti PSII IgG
titer
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.
2
npj Vaccines (2023) 152 Published in partnership with the Sealy Institute for Vaccine Sciences
1234567890():,;

To obtain nanoparticles containing more homogeneous RNA,
we modified 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
45
. The
devised genetic strategy is described in Fig. 3. We named the
resulting VLPs, Qβhp
46
, 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 defined 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
final yield (>90% purity by SEC-HPLC, 16 nm radius by DLS,
2–3 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
time.
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, confirming
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% confidence
interval is indicated by the whiskers. IgG titers between the two
groups were compared by Mann–Whitney 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% Confidence
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 Mann–Whitney 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.
3
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.
DISCUSSION
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 efficient recognition and presentation
to specific surface receptors of immune cells
23,44,47,48
. Several
vaccines based on NPs and VLPs have been developed for delivery
of viral peptide and protein antigens from flu
24,25
, HIV
26
, RSV
27
,
and SARS CoV-2
28–30
. Surface decoration of nanoparticles with
protein antigens has also shown promise for application to
bacterial vaccines
31–34
. 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-specific IgG
responses that protected mice against infectious challenge
35
.
More recently, a modular biosynthetic approach to produce
nanoconjugate vaccines using the SpyTag/SpyCatcher system was
applied for the direct coupling of Shigella flexneri 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
efficient adaptive immune response and prophylaxis against
bacterial challenge
36
.
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-specific antibody func-
tional responses higher than the reference CRM conjugate,
confirming 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
49
. The Qβconjugate
elicited post-1 responses higher than the other nanoconjugates
and like those obtained after two doses of PSII-CRM, confirming
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 confirmed 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 Alzheimer’s disease
and are safe and highly immunogenic
43,50–54
. Further, this study
confirm 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
55
.
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.
4
npj Vaccines (2023) 152 Published in partnership with the Sealy Institute for Vaccine Sciences

Encapsulated RNA represents for about 20–30% of the QβVLP
mass and mainly consists of hairpin-rich molecules derived from
the prophage or encoded in the bacterial host genome
56,57
,
resulting in fragments of highly variable size and composition.
Rhee and colleagues demonstrated that RNA encoding the green
fluorescent protein (GFP) could be delivered into QβVLPs by
adding a hairpin sequence to the end of its coding gene
58
.To
achieve a more reproducible RNA profile 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 profile, mainly
represented by a single major peak encoding the Qβcoat protein,
the new Qβhp
45,59,60
.
Like its precursor, PSII-Qβ, the PSII-Qβhp conjugate induced
high IgG and OPKA titers after a single vaccine dose. To confirm
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)
61
.A
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
62
.
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.
METHODS
Expression and purification of nanoparticles
The genes encoding for ferritin, mI3 and Qβwere synthesized as
DNA strings by GeneArt (Thermo Fisher Scientific) optimizing the
codon usage for expression in the E. coli. The genes were cloned
into pET15b+TEV and pET21b+(Merck-Sigma) PCR-amplified
vectors using the Infusion cloning kit (Takara) following manu-
facturer instructions
33,63
. The plasmid pET24b+encoding the
Qβhp coat protein was purchased by GeneArt (Thermo Fisher
Scientific).
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
4
×7H
2
O 0.5 g/L; KH
2
PO
4
5 g/L; K
2
HPO
4
20 g/L; KOH 1 M to pH final
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
3
PO
4
when needed), 400–800
RPM, air flow rate between 1–2 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
590
value of 5 was reached,
Induction Solution (Glycerol 30 g/l; CaCl
2
0.12 g/L; MgSO
4
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
modified medium (Glycerol 30 g/L; NH
4
Cl 3 g/L; KH
2
PO
4
14 g/L;
K
2
HPO
4
6 g/L; MgSO
4
0.24 g/L; Riboflavin 0.002 g/L; Thiamine
0.002 g/L; Pyridoxine hydrochloride 0.002 g/L; Nicotinamide
0.002 g/L; FeSO
4
1.53 mg/L; MnCl
2
2.64 mg/L; CuSO
4
1.98 mg/L;
ZnSO
4
0.34 mg/L; Na
2
MoO
4
0.6 mg/L); when an OD
590
value of 3
was reached, induction solution (Glycerol 30 g/l; CaCl
2
0.12 g/L;
MgSO
4
0.15 g/L; (NH
4
)
2
SO
4
6 g/L; FeSO
4
9.18 mg/L; MnCl
2
15.84 mg/L; CuSO
4
6.78 mg/L; ZnSO
4
11.88 mg/L; Na
2
MoO
4
2.04 mg/L; IPTG 1 mM) was added and the biomass recovered.
For mI3 purification, bacterial cells were chemically lysed with
CelLytic Express (SIGMA), followed by several steps of purification,
in sequence: Affinity Chromatography Hi Trap HP, Ni
2+
(Cytiva),
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.
Quantification 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(first 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.
5
Published in partnership with the Sealy Institute for Vaccine Sciences npj Vaccines (2023) 152

NaH
2
PO
4
pH7,6 at −20 °C until further use. For Qβand Qβhp
purification, bacterial cells were subjected to mechanical lysis, and
the supernatant flocculated with Polyethyleneimine (PEI). After
centrifugation, the flocculate containing Qβor Qβhp was purified
by Ionic Exchange Chromatography (Cytiva), followed by Size
Exclusion Chromatography Sephacryl S500 (Cytiva) in PBS; the
purified VLPs were stored at −20 °C until further use. Protein
concentration was determined by Pierce™Rapid Gold BCA Protein
Assay Kit (Thermo Fisher Scientific, Illinois, USA) according to
manufacturer’s instructions, using Bovine Serum Albumin (BSA) as
standard.
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
fluorescence 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,
Qβ
PSII-Qβ
0 4 8 12 16 20 24 28 32 36
0
100.000
200.000
300.000
400.000
500.000
600.000
700.000
min
counts
SE-HPLC analysis
PSII-Qβhp
conjugate
Qβhp
PSII-Qβhp
Qβhp
TEMTEM
0 4 8 12 16 20 24 28 32 36
0
200.000
400.000
600.000
800.000
1.000.000
min
counts
PSII-Qβ
conjugate
Qβ
SE-HPLC analysis
aa
bb
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% Confidence 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 Kruskal–Wallis 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.
6
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 purified 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 manufacturer’s instructions. Total RNA was quantified by
Quant-iT™RiboGreen®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; fluores-
cence intensity (Excitation/Emission 485 nm/530 nm) was mea-
sured (Infinite 200, Tecan). RNA total content was expressed as µg/
mL or % RNA/Protein.
For capillary electrophoresis analysis, 20 mL of purified RNA
samples were loaded onto LabCHip GX II (PerkinElmer) following
manufacturer’s instructions. Each sample was analyzed in
triplicate.
For sequencing analysis, three RNA libraries were prepared from
each sample starting with 100 ng of RNA (quantified 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
37
. 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 purified 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 final 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 purified by tangential flow filtration (Sartocon
100 kDa filter). 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 purified ribonucleases, RNase A and RNase T1. After an
overnight incubation at room temperature, RNAses and nucleic
acids were eliminated by serial centrifugal filtration (100 kDa).
Saccharide quantification in the conjugates was performed by
high-performance anion-exchange chromatography with pulsed
amperometric detection (HPAEC-PAD). GBS PSII or PSIa standard
samples at five 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% confidence interval is indicated by the whiskers.
For GMT, non responder sera were assigned titers half of the LLOQ. The Kruskal–Wallis 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.
7
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 trifluoroacetic
acid (Supelco), incubated at 100 °C for 3 h, dried under vacuum
(SpeedVac Thermo), suspended in water, and filtered with 0.45 μm
Phenex-NY (Phenomenex) filters. 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 effluent 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)
6–8 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
64
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 fluorescence 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)
65
. 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 fluorescent OPKA was adapted from
literature
66,67
to increase the throughput capacity to 384-wells
and to avoid CFU counting by introducing a fluorescent dye
(Alamar Blue) that allow to measure the viability endpoint of
bacteria through a plate reader. The fluorescent OPKA used the
same assay components and titration method. The lower limit of
detection was 1:30 dilution and the assay coefficient of variation
was ~30% for both assay formats.
DATA AVAILABILITY
The authors declare that data supporting the findings of this study are available
within the paper and its supplementary information files. All relevant data are
available from the authors.
Received: 23 June 2023; Accepted: 15 September 2023;
REFERENCES
1. Russell, N. J. et al. Risk of early-onset neonatal group B streptococcal disease with
maternal colonization worldwide: systematic review and meta-analyses. Clin.
Infect. Dis. 65, S152–S159 (2017).
2. Lawn, J. E. et al. Group B streptococcal disease worldwide for pregnant women,
stillbirths, and children: why, what, and how to undertake estimates? Clin. Infect.
Dis. 65, S89–S99 (2017).
3. Madrid, L. et al. Infant group B streptococcal disease incidence and serotypes
worldwide: systematic review and meta-analyses. Clin. Infect. Dis. 65, S160–S172
(2017).
4. Schrag, S. J. & Verani, J. R. Intrapartum antibiotic prophylaxis for the prevention of
perinatal group B streptococcal disease: Experience in the United States and impli-
cations for a potential group B streptococcal vaccine. Vaccine 31,D20–D26 (2013).
5. Kobayashi, M. et al. WHO consultation on group B Streptococcus vaccine devel-
opment: Report from a meeting held on 27–28 April 2016. Vaccine 37, 7307–7314
(2019).
6. Nishihara, Y., Dangor, Z., French, N., Madhi, S. & Heyderman, R. Challenges in
reducing group B Streptococcus disease in African settings. Arch. Dis. Child. 102,
72–77 (2017).
7. Cieslewicz, M. J. et al. Structural and genetic diversity of group B Streptococcus
capsular polysaccharides. Infect. Immun. 73, 3096–3103 (2005).
8. Berti, F. et al. Structure of the type IX group B Streptococcus capsular poly-
saccharide and its evolutionary relationship with types V and VII. J. Biol. Chem.
289, 23437–23448 (2014).
9. Paoletti, L. C. et al. Neonatal mouse protection against infection with multiple
group B streptococcal (GBS) serotypes by maternal immunization with a tetra-
valent GBS polysaccharide-tetanus toxoid conjugate vaccine. Infect. Immun. 62,
3236–3243 (1994).
10. Baker, C. J. & Kasper, D. L. Correlation of maternal antibody deficiency with
susceptibility to neonatal group B streptococcal infection. N. Engl. J. Med. 294,
753–756 (1976).
11. Baker, C. J. Group B streptococcal conjugate vaccines. Arch. Dis. Child. 88,
375–378 (2003).
12. Fabbrini, M. et al. Functional activity of maternal and cord antibodies elicited by
an investigational group B Streptococcus trivalent glycoconjugate vaccine in
pregnant women. J. Infect. 76, 449–456 (2018).
13. Paoletti, L. C. et al. Effects of alum adjuvant or a booster dose on immunogen-
icityduring clinical trials of group B streptococcal type III conjugate vaccines.
Infect. Immun. 70, 426–426 (2002).
14. Madhi, S. A. et al. Safety and immunogenicity of an investigational maternal
trivalent group B Streptococcus vaccine in healthy women and their infants: a
randomised phase 1b/2 trial. Lancet Infect. Dis. 16, 923–934 (2016).
15. Madhi, S. A. et al. Antibody kinetics and response to routine vaccinations in
infants born to women who received an investigational trivalent group B
Streptococcus polysaccharide CRM197-conjugate vaccine during pregnancy. Clin.
Infect. Dis. 65, 1897–1904 (2017).
16. Beran, J. et al. Safety and immunogenicity of fully liquid and lyophilized for-
mulations of an investigational trivalent group B Streptococcus vaccine in heal-
thy non-pregnant women: results from a randomized comparative phase II trial.
Vaccine 38, 3227–3234 (2020).
17. Absalon, J. et al. Safety and immunogenicity of a novel hexavalent group B
Streptococcus conjugate vaccine in healthy, non-pregnant adults: a phase 1/2,
randomised, placebo-controlled, observer-blinded, dose-escalation trial. Lancet
Infect. Dis. 21, 263–274 (2021).
F. Carboni et al.
8
npj Vaccines (2023) 152 Published in partnership with the Sealy Institute for Vaccine Sciences

18. Leroux-Roels, G. et al. A randomized, observer-blind phase Ib study to identify
formulations and vaccine schedules of a trivalent Group B Streptococcus vaccine
for use in non-pregnant and pregnant women. Vaccine 34, 1786–1791 (2016).
19. Leroux-Roels, G. et al. Safety and immunogenicity of a second dose of an
investigational maternal trivalent group B Streptococcus vaccine in nonpregnant
women 4–6 years after a first dose: results from a phase 2 trial. Clin. Infect. Dis. 70,
2570–2579 (2020).
20. Margarit, I. et al. Preventing bacterial infections with pilus-based vaccines: the
group B Streptococcus Paradigm. J. Infect. Dis. 199, 108–115 (2009).
21. Stålhammar-Carlemalm, M., Waldemarsson, J., Johnsson, E., Areschoug, T. & Lin-
dahl, G. Nonimmunodominant regions are effective as building blocks in a
streptococcal fusion protein vaccine. Cell Host Microbe 2, 427–434 (2007).
22. Gonzalez-Miro, M. et al. Safety and immunogenicity of the group B Streptococcus
vaccine AlpN in a placebo-controlled double-blind phase 1 trial. iScience 26,
106261 (2023).
23. López-Sagaseta, J., Malito, E., Rappuoli, R. & Bottomley, M. J. Self-assembling
protein nanoparticles in the design of vaccines. Comput. Struct. Biotechnol. J. 14,
58–68 (2016).
24. Kang, S.-M., Kim, M.-C. & Compans, R. W. Virus-like particles as universal influenza
vaccines. Expert Rev. Vaccines 11, 995–1007 (2012).
25. Kanekiyo, M. et al. Self-assembling influenza nanoparticle vaccines elicit broadly
neutralizing H1N1 antibodies. Nature 499, 102–106 (2013).
26. He, L. et al. Presenting native-like trimeric HIV-1 antigens with self-assembling
nanoparticles. Nat. Commun. 7, 12041 (2016).
27. Marcandalli, J. et al. Induction of potent neutralizing antibody responses by a
designed protein nanoparticle vaccine for respiratory syncytial virus. Cell 176,
1420–1431.e17 (2019).
28. Walls, A. C. et al. Elicitation of potent neutralizing antibody responses by designed
protein nanoparticle vaccines for SARS-CoV-2. Cell 183,1367–1382.e17 (2020).
29. Zhang, B. et al. A platform incorporating trimeric antigens into self-assembling
nanoparticles reveals SARS-CoV-2-spike nanoparticles to elicit substantially
higher neutralizing responses than spike alone. Sci. Rep. 10, 18149 (2020).
30. Fougeroux, C. et al. Capsid-like particles decorated with the SARS-CoV-2 receptor-
binding domain elicit strong virus neutralization activity. Nat. Commun. 12, 324
(2021).
31. Wang, L., Xing, D., Le Van, A., Jerse, A. E. & Wang, S. Structure‐based design of
ferritin nanoparticle immunogens displaying antigenic loops of Neisseria
gonorrhoeae. FEBS Open Bio 7, 1196–1207 (2017).
32. Aston-Deaville, S. et al. An assessment of the use of Hepatitis B Virus core protein
virus-like particles to display heterologous antigens from Neisseria meningitidis.
Vaccine 38, 3201–3209 (2020).
33. Cappelli, L. et al. Self-asse mbling protein nanoparticles and virus like particles
correctly display β-barrel from meningococcal factor H-binding protein through
genetic fusion. PLoS ONE 17, e0273322 (2022).
34. Veggi, D. et al. Effective multivalent oriented presentation of meningococcal
NadA antigen trimers by self-assembling ferritin nanoparticles. Int. J. Mol. Sci. 24,
6183 (2023).
35. Polonskaya, Z. et al. T cells control the generation of nanomolar-affinity anti-
glycan antibodies. J. Clin. Invest. 127, 1491–1504 (2017).
36. Li, X. et al. Orthogonal modular biosynthesis of nanoscale conjugate vaccines for
vaccination against infection. Nano Res. 15, 1645–1653 (2022).
37. Wessels, M. R. et al. Immunogenicity in animals of a polysaccharide-protein
conjugate vaccine against type III group B Streptococcus. J. Clin. Invest. 86,
1428–1433 (1990).
38. Paoletti, L. C., Kennedy, R. C., Chanh, T. C. & Kasper, D. L. Immunogenicity of group
B Streptococcus type III polysaccharide-tetanus toxoid vaccine in baboons. Infect.
Immun. 64, 677–679 (1996).
39. Rodrigues, M. Q., Alves, P. M. & Roldão, A. Functionalizing ferritin nanoparticles for
vaccine development. Pharmaceutics 13, 1621 (2021).
40. Hsia, Y. et al. Design of a hyperstable 60-subunit protein icosahedron. Nature 535,
136–139 (2016).
41. Chang, J., Gorzelnik, K. V., Thongchol, J. & Zhang, J. Structural assembly of Qβ
virion and its diverse forms of virus-like particles. Viruses 14, 225 (2022).
42. Akache, B. et al. Anti-IgE Qb-VLP conjugate vaccine self-adjuvants through acti-
vation of TLR7. Vaccines 4, 3 (2016).
43. Fiedler, J. D., Brown, S. D., Lau, J. L. & Finn, M. G. RNA-directed packaging of
enzymes within virus-like particles. Angew. Chem. Int. Ed. 49, 9648–9651 (2010).
44. Tariq, H., Batool, S., Asif, S., Ali, M. & Abbasi, B. H. Virus-like particles: revolutionary
platforms for developing vaccines against emerging infectious diseases. Front.
Microbiol.12, 790121 (2022).
45. Fang, P., Bowman, J. C., Gómez Ramos, L. M., Hsiao, C. & Williams, L. D. RNA:
packaged and protected by VLPs. RSC Adv. 8, 21399–21406 (2018).
46. Carboni, F., Cozzi, R., Margarit, I., Romagnoli, G. & Romano, M. R. Bacterial
immunization using qbeta hairpin nanoparticle constructs. WO/2023/111826
(2023).
47. Pan, J. & Cui, Z. Self‐assembled nanoparticles: exciting platforms for vaccination.
Biotechnol. J. 15, 2000087 (2020).
48. Kheirollahpour, M., Mehrabi, M., Dounighi, N. M., Mohammadi, M. & Masoudi, A.
Nanoparticles and vaccine development. Pharm. Nanotechnol. 8,6–21 (2020).
49. Micoli, F., Adamo, R. & Costantino, P. Protein carriers for glycoconjugate vaccines:
history, selection criteria, characterization and new trends. Molecules 23, 1451
(2018).
50. Cornuz, J. et al. A vaccine against nicotine for smoking cessation: a randomized
controlled trial. PLoS ONE 3, e2547 (2008).
51. Tissot, A. C. et al. Effect of immunisation against angiotensin II with CYT006-
AngQb on ambulatory blood pressure: a double-blind, randomised, placebo-
controlled phase IIa study. Lancet 371, 821–827 (2008).
52. Bachmann, M. F. & Jennings, G. T. Therapeutic vaccines for chronic diseases:
successes and technical challenges. Philos. Trans. R. Soc. B Biol. Sci. 366,
2815–2822 (2011).
53. Huang, X., Wang, X., Zhang, J., Xia, N. & Zhao, Q. Escherichia coli-derived virus-like
particles in vaccine development. npj Vaccines 2,1–8 (2017).
54. C. Gomes, A., Roesti, E. S., El-Turabi, A. & Bachmann, M. F. Type of RNA packed in
VLPs impacts IgG class switching—implications for an influenza vaccine design.
Vaccines 7, 47 (2019).
55. Hong, S. et al. B cells are the dominant antigen-presenting cells that activate
naive CD4+T cells upon immunization with a virus-derived nanoparticle antigen.
Immunity 49, 695–708.e4 (2018).
56. Hung, P. P., Ling, C. M. & Overby, L. R. Self-assembly of Qβand MS2 phage
particles: possible function of initiation complexes. Science 166, 1638–1640
(1969).
57. Heil, F. et al. Species-specific recognition of single-stranded RNA via toll-like
receptor 7 and 8. Science 303, 1526–1529 (2004).
58. Rhee, J.-K. et al. Colorful virus-like particles: fluorescent protein pack aging by the
Qβcapsid. Biomacromolecules 12, 3977–3981 (2011).
59. Fang, P.-Y. et al. Functional RNAs: combined assembly and packaging in VLPs.
Nucleic Acids Res. 45, 3519–3527 (2017).
60. Horn, W. T. et al. Structural basis of RNA binding discrimination between bac-
teriophages Qβand MS2. Structure 14, 487–495 (2006).
61. Pulendran, B., S. Arunachalam, P. & O’Hagan, D. T. Emerging concepts in the
science of vaccine adjuvants. Nat. Rev. Drug Discov. 20, 454–475 (2021).
62. Phares, T. W. et al. Rhesus macaque and mouse models for down-selecting cir-
cumsporozoite protein based malaria vaccines differ significantly in immuno-
genicity and functional outcomes. Malar. J. 16, 115 (2017).
63. Adamo, R., Carboni, F., Cozzi, R., Margarit, I. & Romano, M. R. Bacterial immuni-
zation using nanoparticle vaccine. WO/2021/250628 (2021).
64. Buffi, G. et al. Novel multiplex immunoassays for quantification of IgG against
group B Streptococcus capsular polysaccharides in human sera. mSphere 4,1–15
(2019).
65. Fabbrini, M. et al. A new flow-cytometry-based opsonophagocytosis assay for the
rapid measurement of functional antibody levels against group B Streptococcus.
J. Immunol. Methods 378,11–19 (2012).
66. Bieging, K. T. et al. Fluorescent multivalent opsonophagocytic assay for mea-
surement of functional antibodies to Streptococcus pneumoniae. Clin. Vaccin.
Immunol. 12, 1238–1242 (2005).
67. Mountzouros, K. T. & Howell, A. P. Detection of complement-mediated antibody-
dependent bactericidal activity in a fluorescence-based serum bactericidal assay
for group B Neisseria meningitidis. J. Clin. Microbiol. 38, 2878–2884 (2000).
ACKNOWLEDGEMENTS
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.
AUTHOR CONTRIBUTIONS
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.
COMPETING INTERESTS
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.
9
Published in partnership with the Sealy Institute for Vaccine Sciences npj Vaccines (2023) 152

ADDITIONAL INFORMATION
Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41541-023-00744-5.
Correspondence and requests for materials should be addressed to Immaculada
Margarit or Maria Rosaria Romano.
Reprints and permission information is available at http://www.nature.com/
reprints
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims
in published maps and institutional affiliations.
Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative
Commons license, and indicate if changes were made. The images or other third party
material in this article are included in the article’s Creative Commons license, unless
indicated otherwise in a credit line to the material. If material is not included in the
article’s Creative Commons license and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly
from the copyright holder. To view a copy of this license, visit http://
creativecommons.org/licenses/by/4.0/.
© The Author(s) 2023
F. Carboni et al.
10
npj Vaccines (2023) 152 Published in partnership with the Sealy Institute for Vaccine Sciences