Article
Immunization with Multiple Virulence Factors Provides
Maternal and Neonatal Protection against Group B
Streptococcus Serotypes
Jie Wang 1,2 , Wenbo Li 1,2 , Ning Li 1 and Beinan Wang 1, *
Citation: Wang, J.; Li, W.; Li, N.;
Wang, B. Immunization with
Multiple Virulence Factors Provides
Maternal and Neonatal Protection
against Group B Streptococcus
Serotypes. Vaccines 2023, 11, 1459.
https://doi.org/10.3390/
vaccines11091459
Academic Editor: Ger Rijkers
Received: 2 August 2023
Revised: 24 August 2023
Accepted: 28 August 2023
Published: 5 September 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Vaccines 2023, 11, 1459. https://doi.org/10.3390/vaccines11091459
1 Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of
Sciences, Beijing 100101, China
2 Beijing Varnotech Biopharm Ltd., Beijing 100176, China
* Correspondence: wangbn@im.ac.cn
Abstract: Group B streptococcus (GBS) commonly colonizes the vaginal tract and is a leading cause of
life-threatening neonatal infections and adverse pregnancy outcomes. No effective vaccine is clinically
available. Conserved bacterial virulence factors, including those of GBS, have been employed as
vaccine components. We investigated serotype-independent protection against GBS by intranasal
immunization with six conserved GBS virulence factors (GBSV6). GBSV6 induced systemic and
vaginal antibodies and T cell responses in mice. The immunity reduced mouse mortality and vaginal
colonization by various GBS serotypes and protected newborn mice of immunized dams against GBS
challenge. Intranasal GBSV6 immunization also provided long-lasting protective immunity and had
advantages over intramuscular GBSV6 immunization regarding restricting vaginal GBS colonization.
Our findings indicate that intranasal immunization targeting multiple conserved GBS virulence
factors induces serotype-independent immunity, which protects against GBS infection systemically
and vaginally in dams and prevents newborn death. The study presents valuable strategies for GBS
vaccine development.
Keywords: group B streptococcus; virulence factors; intranasal immunization; vaginal colonization;
maternal vaccines
1. Introduction
Streptococcus agalactiae, or group B streptococcus (GBS), is an encapsulated Gram-
positive opportunistic pathogen that can be classified into 10 different serotypes (Ia, Ib,
II–IX) based on the antigenic and structural properties of the capsular polysaccharide (CPS).
GBS is found in the vagina or lower gastrointestinal tract of about 10–40% of women [1] and
can cause chorioamnionitis, preterm birth, and stillbirth [2,3], as well as neonatal pneumo-
nia, sepsis, and meningitis with early-onset disease (EOD) or late-onset disease (LOD) due
to ascending infection or transmission during delivery [4]. GBS is also responsible for mor-
bidity and mortality in gravidas, the elderly, and immunocompromised adults. The only
available treatment, intrapartum antibiotic prophylaxis (IAP), has substantially reduced
the incidence of EOD. However, in some developing countries where IAP has not been
implemented, EOD incidence is still a leading cause of neonatal mortality. Additionally,
LOD, preterm birth, and stillbirth are not preventable by IAP, remaining a public health
problem in both high- and low/middle-income countries [5]. Hence, improved preventive
measures are urgently needed to fully address the burden of GBS disease for gravidas
and infants.
Maternal vaccines, which lead to placental transfer of GBS antibodies, protect against
multiple outcomes of GBS infection [6]. Currently, there are no approved GBS vaccines
available. Two major types of GBS vaccine have been developed: CPS–protein conjugate
vaccines and protein-based vaccines. CPS–protein conjugate vaccines have shown the most
https://www.mdpi.com/journal/vaccines
Vaccines 2023, 11, 1459 2 of 18

promising results to date in clinical studies [4]. However, there are some concerns regarding
serotype replacement/capsular switching, lack of coverage of non-typeable isolates, and
reduced efficacy owing to the diverse serotype distribution across and within geographic
regions [7]. Protein-based vaccines may confer broad protection across all GBS serotypes
and may also alleviate concerns regarding the threat of serotype replacement/capsular
switching [4,8,9]. Several research groups have identified various surface-exposed proteins
of a wide panel of clinical GBS strains and serotypes that are able to induce opsonically
active antibodies, such as recombinant alpha-like protein subunit vaccines (GBS-NN and
AlpN) that are currently under clinical investigation as broad-spectrum vaccines [10,11].
The selection of optimal bacterial antigens is crucial for vaccine efficacy. Promising
antigens are: (1) surface-exposed or secreted in order for them to be more accessible
to antibodies, (2) conserved across a wide range of serotypes so that they can confer
cross-serotype protection, (3) highly immunogenic in order for them to induce T and B
cell responses, and (4) able to induce long-lasting protective immunity [12,13]. There is
increased awareness of the involvement of GBS protein antigens not only in immunity but
also in pathogenicity [14]. Clinical GBS strains have evolved a wide variety of virulence
factors [15], including adhesins that mediate GBS colonization of the vaginal tract, enabling
transmission to newborns to cause EOD [16]. Other virulence factors, such as FbsB and
ScpB, can facilitate GBS invasion of various tissues, damaging the tissue structure, which
leads to dysfunction of human organs [17,18]. In gaining access to the blood circulation,
Srr2 can help GBS to invade the brain epithelium, causing meningitis [19]. These virulence
factors induce protective immunity in mouse models of infection when used individually
as antigens.
However, single-antigen vaccines may provide variable protection in preclinical mod-
els [20], as immunity induced by a single virulence factor might be ineffective against
isolates lacking the target antigen or inadequate due to the presence of other crucial vir-
ulence factors [21]. Thus, including multiple virulence factors involved in various GBS
pathogenic mechanisms in a vaccine would improve protection against GBS diseases after
induction of antibodies. These antibodies would not only facilitate bacterial clearance via
opsonophagocytosis but would also neutralize the pathogenic functions of the virulence
factors during an infection.
In this study, a combination of six GBS virulence factors involved in different steps
of GBS pathogenesis was used as a vaccine (GBSV6) and delivered through the intranasal
(i.n.) route. The six conserved GBS proteins were: (1) sortase A (SrtA), which is necessary
for anchoring cell wall proteins (including many virulence factors) [22]; (2) cell-surface-
associated protein A (CspA), which cleaves CXC chemokines [23]; (3) C5a peptidase (ScpB),
which specifically cleaves complement-activated C5a, abolishing neutrophil recruitment
to the infection site [24]; (4) a secreted fibrinogen-binding protein (FbsB), which enhances
GBS internalization into epithelial cells [25]; (5) fibrinogen-binding protein with serine-rich
repeat 2 (Srr2), which increases the permeability of the blood–brain barrier [26], and (6) an
immunogenic bacterial adhesin (BibA), which specifically binds to C4-binding protein to
resist opsonophagocytic killing by neutrophils [21,27]. These proteins have been reported
to elicit protective antibodies [28–31] or serotype-independent protection [32,33]. Vaginal
mucosal immunity is necessary for efficient local GBS clearance [34]. Mucosal immuniza-
tion through the i.n. route not only stimulates an immune response in the respiratory
tract, but also induces a strong genital/vaginal mucosal immune response [35,36]. We
hypothesized that i.n. immunization with the set of six conserved virulence factors would
ensure the efficacy and coverage of the GBS vaccine. In this study, we showed that i.n.
GBSV6 immunization of mice reduced GBS colonization of the vagina, protected against
lethal infections, and increased the survival of newborn mice at risk of infection with
various serotypes.
Vaccines 2023, 11, 1459 3 of 18

2. Materials and Methods

2.1. Ethics Statement
This study was performed in strict accordance with the recommendations in the
Guide for the Care and Use of Laboratory Animals of the Institute of Microbiology, Chi-
nese Academy of Sciences (IMCAS) Ethics Committee. The protocols were approved by
the Committee on the Ethics of Animal Experiments of IMCAS (permit number: SQIM-
CAS20211128). All animal experiments, including vaccination, inoculation, and sample
collection, were conducted under isoflurane anesthesia, and all efforts were made to mini-
mize the suffering of the animals. The collection and use of human serum samples were
approved by the Ethics Committee of Beijing Children’s Hospital (2018-162). Informed
consent was obtained from the human subjects prior to enrollment in the study.

2.2. Bacterial Strains and Culture Conditions

GBS serotypes Ib, III (a), and III (b) were clinical isolates obtained from Beijing Chil-
dren’s Hospital. All strains were grown in Todd-Hewitt broth (THB; BD Bioscience, San
Jose, CA, USA) or blood agar plates at 37 ◦ C in 5% CO2 . Overnight cultures were pelleted,
washed, and resuspended in PBS and then used for animal challenge studies. CFUs were
verified by plating on blood agar plates.

2.3. Cloning and Expression of Recombinant Proteins

The DNA sequences of SrtA and BibA were amplified from GBS serotype Ib. The
DNA sequences of Srr2, FbsB, ScpB, and CspA were amplified from GBS serotype III (a).
The primers used for the amplification are listed in Table S1. Recombinant SrtA (amino
acids 82 to 247) and Srr2 (amino acids 192 to 543) were cloned into the pET28a vector
as previously described [19,37]. Recombinant FbsB (amino acids 19 to 628) and BibA
(amino acids 34 to 471) were cloned into the pCold-SUMO vector [16,18]. For CspA and
ScpB, mutations were generated to remove their toxic enzymatic activity. ScpB (amino
acids 32 to 1032), with D130A and S512A site mutations, was cloned into the pET-28a
vector [17]. CspA (amino acids 36 to 1076), with D172A and S567A site mutations, was
cloned into the pET-28a vector [29]. DNA sequences of ScpB and CspA were synthesized
by Shanghai Generay Biotech Co., Ltd. All constructs were transformed into Escherichia
coli BL21 (DE3) for expression. Recombinant proteins were purified as previously de-
scribed [18]. Lipopolysaccharide was removed from the purified proteins to <0.1 endotoxin
units (EU)/µg recombinant protein, using a ToxinEraser endotoxin removal kit (Genscript,
USA), following the manufacturer’s protocol.

2.4. Sample Collection and Preparation

The blood samples were collected through the tail vein of mice and the serum was
obtained by centrifugation, and then stored at −20 ◦ C for subsequent analysis. GBS loads
in vaginal lumen were quantified post-challenge, as previously described [34]. Briefly, a
pre-wetted swab was carefully inserted into the vaginal lumen and gently rotated. The
swabs were soaked and serially diluted in PBS with 0.1% bovine serum albumin and
protease inhibitors for CFU counting on plates. Samples of reproductive tract tissue, bone
marrow, and spleen cells were collected as described by Patras et al. [38]. Briefly, mice
were euthanized by exposure to carbon dioxide gas in a rising concentration. The female
reproductive tract (FRT) tissue was taken, diced into small pieces, and digested with
collagenase IV (Roche Life Science, Indianapolis, IN, USA) for 30 min. Then, the FRT
samples were strained to remove larger clumps before laying on Percoll gradients for
lymphocyte isolation. The collected cells were filtered and washed to generate single-cell
suspension. The spleen and bone marrow (BM) were removed and dissociated into single-
cell suspensions in complete RPMI-1640 medium. The preparation was centrifuged, and
the pelleted cells were analyzed by ELISpot assays for antibody-producing B cells and
IL-17A-producing T cells. All the tissues were treated with ammonium-chloride-potassium
lysis buffer to lyse the red blood cells.
Vaccines 2023, 11, 1459 4 of 18

2.5. Enzyme-Linked Immunosorbent Assay (ELISA)

Antigen-specific antibodies in mouse and human sera were measured by standardized
ELISA. Human serum samples were collected from 96 child patients (aged 5–15 years). The
patients had respiratory infections due to various identified or non-identified pathogens. In
brief, Corning Costar 96-well plates (Fisher Scientific, Waltham, MA, USA) were coated with
5 µg/mL of GBSV6 or each of the six proteins at 4 ◦ C overnight. For bacteria coating, surface
protein extraction of GBS was prepared using the protocol described by Hughes et al. [39].
Then, 100 µL/well of the extraction was added to coat the plates. After antigen removal,
the plates were incubated with sealing buffer (200 µL/well of 5% skim milk) at 37 ◦ C for 2 h.
Next, 100 µL of each of the 1:1000 diluted mouse or human serum samples were added to
the plates following washing with PBS buffer three times. After 2 h of incubation at 37 ◦ C,
the serum samples were removed, and the wells were rinsed with PBST buffer six times.
Then, 1:4000 diluted HRP-conjugated goat anti-mouse IgG, or goat anti-mouse IgA antibody
(Southern Biotech, Birmingham, AL, USA), was added. Following 1 h of incubation at 37 ◦ C
and washing with PBST buffer six times, TMB Substrate (Tiangen Biotech, Beijing, China)
was added (50 µL/well) and incubated for 15–30 min at 37 ◦ C under dark conditions. Stop
Solution (50 µL/well) was added to stop the reaction. The ELx800 plate reader (BioTek,
Winooski, VT, USA) was used to measure absorbance at 450 nm, and the absorbance at
630 nm was used as the internal control. A standard curve was generated by adding
double-diluted purified mouse IgG (Alpha Diagnostic Int. Inc., San Antonio, TX, USA)
or IgA (Bethyl Laboratories, Montgomery, TX, USA) to anti-mouse IgG- or anti-mouse
IgA-coated wells. The concentration of the antibody levels was calculated according to the
standard curve.

2.6. Enzyme-Linked Immunospot (ELISpot) Assays

ELISpot assays of T and B cells were conducted as previously described [40]. Antigen-
specific IL-17A-secreting cells or IFN-γ-secreting cells were detected with kits of Mouse
IL-17A ELISPOT BASIC or Mouse IFN-γ ELISPOT BASIC (Mabtech, Minneapolis, MN,
USA) according to the manufacturer’s instructions. Briefly, the PVDF membranes were
pretreated with 35% ethanol, and the plates were coated with mouse anti-IL-17-I or AN18
for 24 h. Single-cell suspensions of the FRTs, bone marrow, or spleen cells (4 × 105 ) were
added with or without 2.0 µg/100 ul of PBS of GBSV6, or 2.0 ug each of the six proteins
of GBSV6 in 100 ul of PBS overnight. Plates were washed and incubated with 100 µL of
0.25 µg/mL of biotinylated monoclonal antibody MT2270 or 1 µg/mL of R4-6A2, followed
by Streptavidin–HRP. The plates were developed using established procedures by 3-amino-
9-ethylcarbazole (AEC) (Millipore, Bedford, MA, USA). Spots were enumerated with an
ImmunoSpot Analyzer (Cellular Technology Ltd., Beachwood, OH, USA) and multiplied
by the dilution factor.

2.7. GBS Opsonophagocytic Assay

The opsonophagocytic killing assay (OPKA) was conducted with HL-60 cells as
described previously [41], with modifications. The HL-60 cell line is a promyelocytic
cell line derived from human leukemia. The cells can be induced to differentiate into
granulocyte-like cells, or neutrophils, and widely used for OPKA to measure the efficacy
of bacterial vaccines. Briefly, HL-60 cells were differentiated into neutrophil-like cells
by culturing in RPMI medium (Invitrogen, Life Technologies Ltd., CA, USA) containing
0.8% N, N-dimethylformamide (DMF) for 5–7 days. OPKA reactions were performed
in 96-well plates with heat-inactivated (56 ◦ C for 30 min) serum from immunized mice,
1 × 103 CFU of GBS in opsonization buffer, 2 × 106 differentiated HL-60 cells, and 10% baby
rabbit complement (Pel-Freez, Rogers, AR, USA). Control reactions were performed with
non-immune serum or without GBS cells. The mixtures were incubated on a mini-orbital
shaker (700 rpm) for 45 min at 37 ◦ C in a 5% CO2 atmosphere. The reaction mixture from
each well was collected, serially diluted, and plated on blood agar plates. The experiment
was performed in triplicate. CFUs were counted after leaving overnight at 37 ◦ C in a
Vaccines 2023, 11, 1459 5 of 18

5% CO2 atmosphere. The percentage of killed bacteria was determined by comparing the
CFUs in tests carried out without effector cells (100% surveillance) to those of the tested
samples, subtracting the percent that survived from 100%.

2.8. GBS Adherence Assays

The adherence assay was performed as previously described [42]. A549 cells (human
lung carcinoma cells, ATCC CCL185) were grown to confluency in 24-well plates overnight.
GBS (3 × 106 ) was mixed with anti-GBSV6 serum or serum from CpG control mice (1:1, v/v)
and incubated for 30 min. Then, samples were added to A549 cells at a multiplicity of
infection of 10 and incubated for 2 h. Cells were washed with PBS 6 times and 0.1% Triton
X-100 was added to lyse the cells. Lysate was plated on selective blood agar plates to
enumerate bacterial CFUs. The adherent CFU percentage was calculated as: [(recovered
CFU)/(original inoculum CFU)] × 100%.

2.9. GBS Invasion Assay

The human brain microvascular endothelial cell line (hBMEC) was kindly provided by
Prof. Xiang-Ru Wang from Huazhong Agricultural University, Wuhan, Hubei, China, and
cultured as described by Yang et al. [43]. The bacterial invasion assay was performed using
hBMEC, as previously described [44]. Briefly, GBS was grown overnight in THB and then
added to confluent hBMEC monolayers at a multiplicity of infection of 0.1 (1 × 104 CFU).
Then, 30 min after infection, monolayers were treated with a penicillin and streptomycin
mixture (100 µg/mL) for 30 min to remove extracellular GBS, and cells were lysed with
0.1% Triton X-100. Cell lysates were plated on blood agar plates to enumerate the intracel-
lular bacteria. To determine the inhibitory effects of anti-GBSV6 serum on GBS invasion,
250 µL of GBS (2.5 × 105 CFU) was preincubated with 250 µL of mouse immune serum for
30 min at room temperature, and 20 µL of GBS (1 × 104 CFU) was added to the cells. GBS
invasion was calculated as: [(recovered CFU)/(initial inoculum CFU)] × 100%.

2.10. Mice Immunization and Challenge

Specific pathogen-free (SPF) female CD-1 mice (6-week-old; Vital River Laboratory
Animal Center, Beijing, China) were immunized (30 µL) 3 times (days 0, 7, and 14) through
the intranasal (i.n.) or intramuscular (i.m.) route with 60 µg of GBSV6 (10 µg of each
recombinant protein) and 10 µg of CpG-oligodeoxynucleotides (CpG-OND 1826; Generay
Biotechnology, China) [45].
Vaginal colonization and lethal challenge experiments were performed with 10-week-old
SPF female CD-1 mice. For vaginal colonization, a mouse model described by Patras et al. was
employed [38]. In brief, mice were intraperitoneally injected with β-estradiol (0.5 mg/mouse)
and vaginally challenged with GBS serotype Ib or GBS III (a) (1.0 × 107 /10 µL) 2 weeks after
the last immunization. For systemic infection, dose escalation studies were conducted
to determine the lethal challenge dose. Mice were challenged i.v. with 5 × 107 , 1 × 108 ,
and 2 × 108 /mouse, respectively. The survival rates were monitored, whereby 2 × 108 of
GBS serotype Ib or serotype III (a) caused 70–90% death within 10 days after the challenge
and was used as a lethal challenge dose. Mice were challenged with the lethal dose of
GBS serotype Ib or III (a) through the tail vein. Survival was monitored twice per day for
10 days. Mice with a weight loss of 25% of their starting body weight were euthanized and
recorded as dead.
The maternal immunization/newborn challenge experiment was performed as de-
scribed by Buccato et al. [46]. In brief, CD-1 female mice were mated 3 days after the last
immunization. Within 48 h of birth, the newborns were intraperitoneally challenged with
2 × 106 CFUs of GBS serotype Ib or III (which is lethal to 80–90% of newborns). Survival of
the newborns was monitored for 3 days after challenge. Mortality was an anticipated out-
come and was approved by the Animal Ethics Committee. Surviving mice were euthanized
at the end of the experiment.
Vaccines 2023, 11, 1459 6 of 18

2.11. Statistical Analyses

Statistical analyses were performed in GraphPad Prism v7.0. Vaginal CFUs were com-
pared using two-tailed unpaired Mann–Whitney U tests. Normally distributed continuous
variables were compared between two groups using unpaired, two-tailed Student’s t tests.
Normally distributed continuous variables were compared among more than two groups
using one- or two-way analysis of variance, followed by Tukey’s multiple comparisons
tests. The survival and percentage of colonized mice were compared over time between
groups using log-rank (Mantel–Cox) tests. Survival of newborns was compared using
Fisher’s exact test. Statistical significance was defined as p < 0.05.

3. Results
3.1. Immunization i.n. with GBSV6 Induced Antibody Responses in the Blood and Vagina
The virulence factors, including SrtA, ScpB, CspA, FbsB, Srr2, and BibA, play various
roles in GBS pathogenesis and are expressed by highly pathogenic strains, a variety of
clinical isolates, or almost all GBS serotypes. The selected proteins were cloned, expressed,
and purified, and their purity was determined by SDS-PAGE (Figure S1). They were then
pooled (10 µg of each) and designated GBSV6. Mice were immunized with i.n. GBSV6,
along with CpG-oligodeoxynucleotides (CpG) as an adjuvant, in phosphate-buffered saline
(PBS) three times at one-week intervals. Control mice were inoculated with CpG in PBS.
The enzyme-linked immunosorbent assay (ELISA) revealed that GBSV6-specific antibodies
in the serum (IgG) and vaginal lavage fluid (IgA) were induced in the immunized mice
(Figure 1A,B). These serum IgG and vaginal mucosal IgA were directed against each of
the GBSV6 proteins (Figure 1C,D), with strong serum IgG responses to ScpB and CspA
and strong vaginal IgA responses to ScpB. These results indicate that i.n. GBSV6 is highly
immunogenic and induced strong antibody responses both systemically and locally.

3.2. GBSV6-Specific Antibodies Recognized Clinical GBS Isolates

GBSV6 contains surface proteins. We determined whether the GBSV6-specific antibod-
ies could recognize clinical GBS isolates. Sera from mice i.n. immunized with each GBSV6
protein individually had higher levels of bacterial binding to GBS serotypes Ib, III (a), and
III (b) compared to sera from CpG control mice, according to the ELISA (Figure 2A–C).
This indicates that the antibodies induced in GBSV6-immunized mice can recognize native
target molecules on the bacterial surface.

3.3. Antibody Response to GBSV6 in Humans

GBS is a frequent isolate from children. Serum samples from 96 child patients were
collected. These patients had respiratory infections due to various identified or non-
identified pathogens. The ELISA showed that the serum samples bound to clinical GBS
serotypes Ib, III (a), and III (b) (Figure 2D). They also had higher mean responses to each
GBSV6 protein compared to a non-related antigen (keyhole limpet hemocyanin, KLH)
(Figure 2E). These results indicate that human antibodies specifically recognize GBS and
GBSV6 antigens.

3.4. GBSV6 Induced T Cell Responses

The Th17 response to GBSV6 was determined in the female reproductive tracts (FRTs)
and spleen by the T cell enzyme-linked immunospot assay (ELISpot) 2 weeks after the last
immunization. High numbers of IL-17-secreting CD4+ T cells were found in the FRTs and
spleens of GBSV6-immunized mice, while these cells were not detected in the CpG control
mice (Figure 3A,B). Higher numbers of IFN-γ-secreting CD4+ T cells were also detected
in GBSV6-immunized mice compared to CpG control mice (Figure 3C,D). These results
indicate that i.n. GBSV6 immunization induces Th17 and Th1 responses in mucosal and
systemic lymphoid tissues.
Vaccines 2023, 11, x FOR PEER REVIEW 7 of 19

Vaccines 2023, 11, 1459 7 of 18

Figure 1. Intranasal (i.n.) GBSV6 immunization induced antibody responses in the blood and vagina.
Figure 1. Intranasal
Mice were (i.n.) GBSV6
i.n. immunized immunization
3 times at induced
1-week intervals antibody
with GBSV6 responses
or CpG. in the
Two weeks blood
after and
the last
vagina. Mice were i.n. immunized 3 times at 1-week intervals with GBSV6 or CpG. Two weeks
immunization, GBSV6-specific antibodies were measured in (A) serum and (B) vaginal lavage fluid after
thebylast
theimmunization,
enzyme-linkedGBSV6-specific
immunosorbentantibodies were(C)
assay (ELISA). measured in (A)
Serum IgG andserum and (B)
(D) vaginal vaginal
IgA lav-
targeting
ageeach of the GBSV6 antigens. Data represent mean ± standard error of the mean (SEM) based IgA
fluid by the enzyme-linked immunosorbent assay (ELISA). (C) Serum IgG and (D) vaginal on
targeting each of the GBSV6 antigens. Data represent mean ± standard error of the mean (SEM)
two independent experiments (n = 10). * p < 0.05, ** p < 0.01, **** p < 0.0001.
based on two independent experiments (n = 10). * p < 0.05, ** p < 0.01, **** p < 0.0001.
3.5. GBSV6-Specific Antibodies Promoted GBS Opsonophagocytosis and Prevented GBS
Adherence
3.2. to and Invasion
GBSV6‐Specific of Host
Antibodies Cells
Recognized Clinical GBS Isolates
More bacteria
GBSV6 containswere killed
surface when GBS
proteins. We strains were pretreated
determined whether the with sera from GBSV6-
GBSV6-specific anti-
immunized mice than sera from CpG control mice, and the increased opsonophagocytic
bodies could recognize clinical GBS isolates. Sera from mice i.n. immunized with each
killingprotein
GBSV6 was observed for various
individually GBS serotypes,
had higher levels of i.e., Ib, III binding
bacterial (a), and III (b) (Figure
to GBS 4A–C).
serotypes Ib, III
(a), and III (b) compared to sera from CpG control mice, according to the ELISA to
The roles of GBSV6-specific antibodies in the prevention of GBS adherence and
(Figure
invasion of host cells were evaluated using A549 cells (human lung epithelial cells) and
2A–C). This indicates that the antibodies induced in GBSV6-immunized mice can recog-
human brain microvascular endothelial cells (hBMECs), respectively. GBS adherence to
nize native target molecules on the bacterial surface.
A549 cells (Figure 4D,E) or invasion of hBMECs (Figure 4F,G) were significantly reduced
when GBS strains were pretreated with sera from GBSV6-immunized mice compared to
sera from CpG control mice. These results indicate that GBSV6-specific antibodies can im-
pede bacterial attachment to epithelial cells (preventing colonization) and inhibit bacterial
penetration of the host brain endothelium (preventing crossing of the blood–brain barrier).

3.6. GBSV6 Immunization Promoted GBS Clearance in the Vagina and Protected against Systemic
GBS Infection
A mouse model was employed for the vaginal colonization assay. Two weeks after the
last immunization, mice were intraperitoneally injected with β-estradiol to synchronize
the mice into pre-estrus, imitating a condition for GBS colonization in humans. Then, 24 h
later, mice were vaginally challenged with GBS serotype Ib. Colony-forming units (CFUs)
Vaccines 2023, 11, 1459 8 of 18

in vaginal lavage fluid were determined over 35 days following the challenge. CFUs were
lower in GBSV6-immunized mice compared to CpG control mice at all detection time
points (Figure 5A). By 35 days after challenge, the bacteria were completely cleared in all
GBSV6-immunized mice, while up to 103 CFUs were detected in CpG control mice. Similar
results were also observed in mice challenged with GBS serotype III (a) (Figure 5B).
To evaluate the protective role of GBSV6 against systemic infections, GBSV6-immunized
mice were intravenously challenged with a lethal dose of GBS serotype Ib and lethality was
monitored over 10 days. Here, 70% of CpG control mice died on day 1 post-challenge and
the lethality increased to 90% within 2 days. In contrast, only 20% of GBSV6-immunized
mice died on day 1 and no others died thereafter (Figure 5C). This survival assay was also
performed with GBS serotype III (a) challenge. Similar to the results regarding protection
against systemic GBS serotype Ib infection, 10% of CpG control mice survived, while 70% of
GBSV6-immunized mice survived (Figure 5D). These results indicate that i.n. GBSV6
Vaccines 2023, 11, x FOR PEER REVIEW 8 of 19
immunization induces immunity in the reproductive tract and circulation, and it offers
cross-protection against GBS serotypes both locally and systemically.

Figure 2. GBSV6-specific
GBSV6-specific antibodies
antibodies recognized
recognizedGBS GBScells
cellsand
andwere
wereantigenic
antigenicininhumans.
humans.Serum
SerumIgG
IgG
responses
responses to GBS serotypes (A) Ib, (B) III (a), and (C) III (b) in mice immunized with eachGBSV6
to GBS serotypes (A) Ib, (B) III (a), and (C) III (b) in mice immunized with each GBSV6
antigen, determined by
antigen, determined by the
the ELISA. Data represent
ELISA. Data mean ±
represent mean ± SEM
SEM(n (n==5).
5).Human
Humanserum
serumIgG
IgGresponses
responses
to (D) various GBS serotypes and (E) to each of the GBSV6 antigens. Data represent mean ± SEM (n
to (D) various GBS serotypes and (E) to each of the GBSV6 antigens. Data represent mean ± SEM
= 5). Keyhole limpet hemocyanin (KLH) was used as a negative control (n = 96). * p < 0.05, ** p < 0.01,
(n = 5). Keyhole limpet hemocyanin (KLH) was used as a negative control (n = 96). * p < 0.05,
*** p < 0.001, **** p < 0.0001.
** p < 0.01, *** p < 0.001, **** p < 0.0001.
3.3. Antibody Response to GBSV6 in Humans
GBS is a frequent isolate from children. Serum samples from 96 child patients were
collected. These patients had respiratory infections due to various identified or non-iden-
tified pathogens. The ELISA showed that the serum samples bound to clinical GBS sero-
types Ib, III (a), and III (b) (Figure 2D). They also had higher mean responses to each
GBSV6 protein compared to a non-related antigen (keyhole limpet hemocyanin, KLH)
(Figure 2E). These results indicate that human antibodies specifically recognize GBS and
GBSV6 antigens.
 Vaccines 2023, 11, 1459 9 of 18
Vaccines 2023, 11, x FOR PEER REVIEW 9 of 19

Figure 3. T cell3.responses
Figure to GBSV6.
T cell responses toMice wereMice
GBSV6. immunized as describedasindescribed
were immunized Figure 1. in
Two weeks1. Two weeks
Figure
after theafter
last immunization, the FRT and spleen samples were collected. (A) IL-17A- and (C) IFN-γ-
the last immunization, the FRT and spleen samples were collected. (A) IL-17A- and (C) IFN-γ-
secreting T cells in the FRTs, and (B) IL-17A- and (D) IFN-γ-secreting T cells in the spleens, in re-
secreting T cells in the FRTs, and (B) IL-17A- and (D) IFN-γ-secreting T cells in the spleens, in response
sponse to GBSV6, were determined by ELISpot. Data represent mean ± SEM based on two inde-
pendenttoexperiments
GBSV6, were (n =determined by ELISpot. Data represent mean ± SEM based on two independent
10). **** p < 0.0001.
experiments (n = 10). **** p < 0.0001.
3.5. GBSV6‐Specific Antibodies Promoted GBS Opsonophagocytosis and Prevented GBS
3.7. Maternal i.n. GBSV6 Immunization Protected Neonatal Mice against GBS Challenge as
Adherence to and Invasion of Host Cells
Efficiently as Maternal i.m. (Intramuscular) GBSV6 Immunization
More bacteria were killed when GBS strains were pretreated with sera from GBSV6-
We first evaluated the protective effects of GBSV6 in newborns of i.m. immunized
immunized mice than sera from CpG control mice, and the increased opsonophagocytic
dams. Female mice were mated three days following the last i.m. GBSV6/aluminum
killing was observed for various GBS serotypes, i.e., Ib, III (a), and III (b) (Figure 4A–C).
(Alum) immunization or Alum-only inoculation. Their offspring were challenged with GBS
serotype Ib (1 × 106 CFUs/mouse). Less than 7.4% (7/95) of the newborns of the Alum
control dams survived, whereas up to 79% (83 of 105) the newborns of the i.m. immunized
dams survived (Table 1). This indicates that systemic maternal anti-GBSV6 immunity
protects newborns against GBS infection through placental transfer to the fetus. As i.n.
GBSV6 immunization induced high levels of serum GBS-specific antibodies, we expected
protection of newborns of i.n. immunized dams. Similar to the newborns of i.m. immunized
dams, after challenge with the same dose of GBS serotype Ib, up to 85% (91/107) of the
newborns of i.n. immunized dams survived compared to 8.3% (9/108) of the newborns of
CpG control dams (Table 1). The newborns from i.n. immunized dams were also challenged
with GBS serotype III (a) to assess cross-serotype protection. There were significantly
more surviving newborns of GBSV6-immunized dams compared to CpG control dams,
indicating that the newborns acquired protective levels of maternal antibodies from i.n.
immunized dams.
Vaccines 11, 1459
2023,2023,
Vaccines 11, x FOR PEER REVIEW 1010
of of
18 19

Figure 4. GBSV6-specific
Figure 4. GBSV6-specific antibodies
antibodiespromoted
promoted GBS opsonophagocytosis
GBS opsonophagocytosis and prevented
and prevented GBSGBSadher-
adher-
enceence
to and invasion of host cells. GBS serotypes (A) Ib, (B) III (a), and (C) III (b) were
to and invasion of host cells. GBS serotypes (A) Ib, (B) III (a), and (C) III (b) were incubated incubated
withwith
differentiated
differentiatedHL-60 cellscells
HL-60 in the presence
in the of sera
presence from
of sera fromGBSV6-immunized
GBSV6-immunized or CpG
or CpGcontrol mice
control mice
for 45
formin, andand
45 min, thenthen
the the
bacterial growth
bacterial growthraterate
waswas
determined
determined by CFU
by CFU counting. ForFor
counting. thethe
adherence
adherence
assay,
assay, A549A549
cellscells
werewere infected
infected withwithGBSGBS serotype
serotype (D)(D)
Ib orIb(E)
or (E) III (a)
III (a) at aatmultiplicity
a multiplicity of infection
of infection of of
10 for 2 h, washed to remove the non-adhered bacteria, lysed, and
10 for 2 h, washed to remove the non-adhered bacteria, lysed, and plated onto blood agar plates forfor
plated onto blood agar plates
CFUCFU counting.
counting. For For
the the invasion
invasion assay,assay, monolayers
monolayers of human
of human brainbrain microvascular
microvascular endothelial
endothelial cells
cells
(hBMECs) were infected with GBS serotype (F) Ib or (G) III (a) for 30 min, washed and incubated
(hBMECs) were infected with GBS serotype (F) Ib or (G) III (a) for 30 min, washed and incubated
with penicillin and streptomycin for 30 min to remove extracellular bacteria, lysed, and plated onto
withblood
penicillin
agar and
platesstreptomycin for 30 min
for CFU counting. Datatorepresent
remove extracellular
mean ± SEM bacteria,
(n = 5). **lysed, and
p < 0.01, ***plated onto****
p < 0.001,
bloodp <agar plates for CFU counting. Data represent mean ± SEM (n = 5). ** p < 0.01, *** p < 0.001,
0.0001.
**** p < 0.0001.
The roles of GBSV6-specific antibodies in the prevention of GBS adherence to and
3.8. GBSV6 Promoted Clearance of Ascending GBS Infection When Mice Were Immunized through
invasion of the
the i.n. but Not host cells
i.m. were evaluated using A549 cells (human lung epithelial cells) and
Route
human brain microvascular endothelial cells (hBMECs), respectively. GBS adherence to
In addition to the high levels of serum IgG antibodies, the i.n. immunization induced
A549 cells (Figure 4D,E) or invasion of hBMECs (Figure 4F,G) were significantly reduced
GBSV6-specific vaginal IgA (Figure 1B,D). This represents an advantage over i.m. immu-
when GBS strains were pretreated with sera from GBSV6-immunized mice compared to
nization, as vaginal IgA offers efficient protection against GBS colonization of the vagina.
sera from CpG control mice. These results indicate that GBSV6-specific antibodies can im-
Mice were immunized with GBSV6 through the i.n. or i.m. route, and bacterial colonization
pede bacterial attachment to epithelial cells (preventing colonization) and inhibit bacterial
of the vagina was compared after intravaginal challenge with GBS serotype Ib, as before.
penetration of the host brain endothelium (preventing crossing of the blood–brain bar-
After the challenge, CFUs were significantly reduced on days 3, 6, 9, and 15 in the i.n. group
rier).
but not in the i.m. group, while there were no significant CFU reductions in the i.m. group
compared to the Alum control group at any detection time point (Figure 6A). Of the 5 mice
in the i.n. group, 2 had no CFUs by day 21, while up to 104 CFUs were detected in the i.m.
 A mouse model was employed for the vaginal colonization assay. Two weeks after
the last immunization, mice were intraperitoneally injected with β-estradiol to synchro-
nize the mice into pre-estrus, imitating a condition for GBS colonization in humans. Then,
24 h later, mice were vaginally challenged with GBS serotype Ib. Colony-forming units
Vaccines 2023, 11, 1459 11 of 18
(CFUs) in vaginal lavage fluid were determined over 35 days following the challenge.
CFUs were lower in GBSV6-immunized mice compared to CpG control mice at all detec-
tion time points (Figure 5A). By 35 days after challenge, the bacteria were completely
mice. By in
cleared 35 all
days following the challenge,
GBSV6-immunized mice, no CFUs
while upwere
to 10found
3 CFUsinwere
any mice in theini.n.
detected CpGgroup,
control
while 2 of the 5 mice in the i.m. group still had CFUs. Similar results were also observed
mice. Similar results were also observed in mice challenged with GBS serotype III (a) (Fig-
after challenge with GBS serotype III (a) in the i.n. and i.m. groups (Figure 6B). These
ure 5B).
results indicate that vaginal GBS is cleared faster in the i.n. group than the i.m. group.

Figure5.5.GBSV6
Figure GBSV6protected
protected against
against vaginal
vaginal andand systemic
systemic infections
infections by bothby GBS
bothserotype
GBS serotype Ib (a).
Ib and III and III
(a). Mice were immunized as described in Figure 1. Two weeks after the last immunization, the mice
Mice were immunized as described in Figure 1. Two weeks after the last immunization, the mice
were intravaginally challenged with 1 × 107 CFUs of GBS serotype (A) Ib or (B) III (a) and then CFUs
were intravaginally challenged with 1 × 107 CFUs of GBS serotype (A) Ib or (B) III (a) and then CFUs
in vaginal lavage fluid were counted at the indicated time points over the following 35 days. Data
in vaginal lavage fluid were counted at the indicated time points over the following 35 days. Data
represent mean ± SEM (n = 5). Two weeks after the last immunization, the mice were administered
represent meanof±GBS
a lethal dose SEMserotype
(n = 5). Two weeks
(C) Ib after
or (D) III the last immunization,
(a) through the and
the tail vein, micethe
were administered
mortality of theamice
lethal dose of GBS
was recorded serotype
for 10 days (n(C) Ib or* (D)
= 10). III (a)**through
p < 0.05, p < 0.01.the tail vein, and the mortality of the mice
was recorded for 10 days (n = 10). * p < 0.05, ** p < 0.01.
ToGBSV6
Table 1. evaluate the protective
protected roleagainst
neonatal mice of GBSV6
GBS against systemic infections, GBSV6-immun-
challenge.
ized mice were intravenously challenged with a lethal dose of GBS serotype Ib and lethal-
Immunization
ity was monitoredRoute,
over 10GBS
days. Here, 70% No. of Mice
of CpG Protected
control mice died on day 1Ratio
post-chal-
Strains Protection
Vaccine /No. of Mice Treated
lenge and the lethality increased to 90% within 2 days. In contrast, only 20% of GBSV6-
Intranasal
immunized mice died on day 1 and
GBS Ib no others died thereafter (Figure 5C). This survival
9/108
assay was CpG
also performed with GBS serotype III (a) challenge. Similar to the results re-
GBSV6 GBS Ib 91/107 83.7 *
garding protection
CpG
against GBS
systemic
III (a)
GBS serotype15/101
Ib infection, 10% of CpG control mice
survived, while 70% of GBSV6-immunized
GBSV6 GBS III (a) mice survived
77/98 (Figure 5D). These74.8 *results indi-
cate that i.n. GBSV6 immunization induces immunity in the reproductive tract and circu-
Intramuscular
GBS Ib 7/95
Alum
lation, and it offers cross-protection against GBS serotypes both locally and systemically.
GBSV6 GBS Ib 83/105 77.4 *
* Female mice were intranasally (i.n.) immunized with GBSV6/CpG or i.m. with GBSV6/Alum, as described
in Figure 1. Control mice were inoculated with CpG or Alum alone. Mice were mated 3 days after the last
immunization. The offspring were intraperitoneally challenged with GBS serotype Ib or III (a) (2 × 106 ) within
48 h of birth. Survival of neonatal mice was monitored for 3 days after challenge. Protection values were calculated
as follows: [(% dead in control group-% dead in vaccine group)/% dead in control group] × 100. Data are from
two independent experiments. * p < 0.05 (Fisher’s exact test).
Vaccines 2023, 11, x FOR PEER REVIEW 13 of 19

Vaccines 2023, 11, 1459 (Figure 6B). These results indicate that vaginal GBS is cleared faster in the i.n. group than 12 of 18
the i.m. group.

Figure 6. GBSV6
Figure promoted
6. GBSV6 clearance
promoted of ascending
clearance GBS infection
of ascending when when
GBS infection mice were immunized
mice were immunized though
though the
the i.n
i.nbut
butnot
notthe thei.m.
i.m.route.
route.Mice
Micewere
werei.n.
i.n.immunized
immunizedwith with GBSV6/CpG or i.m.
GBSV6/CpG or i.m. with withGBSV6/Alum
GBSV6/Alum 3 times at 1-week intervals. Control groups were inoculated i.n. with CpG or i.m. with
3 times at 1-week intervals. Control groups were inoculated i.n. with CpG or i.m. with Alum.
Alum. Two weeks after the last immunization, the mice were intravaginally challenged with GBS
serotype (A) weeks
Two after
Ib or (B) theand
III (a) lastCFUs
immunization, thelavage
in the vaginal mice were
fluid intravaginally
were determinedchallenged with GBS serotype
at the indicated
time points after challenge. Data represent mean ± SEM (n = 5). Two weeks after the last immuniza- time points
(A) Ib or (B) III (a) and CFUs in the vaginal lavage fluid were determined at the indicated
after challenge.
tion, GBSV6-specific (C) Data
serumrepresent mean ±IgG,
IgG, (D) vaginal SEM(E)(nserum
= 5). Two
IgA, weeks
and (F)after theIgA
vaginal last antibodies
immunization, GBSV6-
in serum and vaginal lavage fluid were measured by the ELISA. Data are from two independent
specific (C) serum IgG, (D) vaginal IgG, (E) serum IgA, and (F) vaginal IgA antibodies in serum
experiments (n = 10).lavage
and vaginal * p < 0.05, ***were
fluid p < 0.001, **** p <by
measured 0.0001.
the ELISA. Data are from two independent experiments
(n = 10). * p < 0.05, *** p < 0.001, **** p < 0.0001.

Antibodies in the serum and vaginal lavage fluid were compared between the i.n.
and i.m. groups. The levels of GBSV6-specific IgG in the serum and vaginal lavage
fluid samples were similar between the two groups (Figure 6C,D). Serum IgA in the i.m.
group was induced but much lower compared to the i.n. group (Figure 6E). A robust
IgA response in the vagina was induced in the i.n. group but was undetectable in the
i.m. group (Figure 6F). These observations indicate that the vaginal IgA response induced
 Antibodies in the serum and vaginal lavage fluid were compared between the i.n.
and i.m. groups. The levels of GBSV6-specific IgG in the serum and vaginal lavage fluid
Vaccines 2023, 11, 1459
samples were similar between the two groups (Figure 6C,D). Serum IgA in the i.m. group13 of 18
was induced but much lower compared to the i.n. group (Figure 6E). A robust IgA re-
sponse in the vagina was induced in the i.n. group but was undetectable in the i.m. group
(Figure 6F). These observations indicate that the vaginal IgA response induced by immun-
by immunization
ization through thethrough
i.n. routethe i.n. route
is required forisefficient
required for efficient
protection protection
against against GBS
GBS colonization
colonization of the tract.
of the reproductive reproductive tract.

3.9. GBSV6Induced
3.9. GBSV6 InducedLong‐Lasting
Long-Lasting Plasma
Plasma Cells
Cells andand Th17
Th17 Cells
Cells and and Provided
Provided Efficient
Efficient
Long-Term Protection
Long‐Term Protection
To determine
To determinewhether
whetheri.n.i.n. immunization
immunization cancan induce
induce long-lasting
long-lasting immunity,
immunity, antibody
antibody
and T cell responses were examined six months after the last immunization.
and T cell responses were examined six months after the last immunization. The ELISA The ELISA
revealed
revealed that i.n. immunized mice maintained 700 ng/mL of serum IgG, although the level level
that i.n. immunized mice maintained 700 ng/mL of serum IgG, although the
was lowercompared
was lower comparedtoto the
the level
level at 2atweeks
2 weeks
afterafter immunization
immunization (Figure (Figure 7A). Compared
7A). Compared to
to 1 ng/mL
1 ng/mL in the
in the CpGCpG control
control group,
group, the vaginal
the vaginal IgA level
IgA level in i.n.inimmunized
i.n. immunized
mice atmice
6 at
6months
months waswas
246246 ng/mL
ng/mL (range,
(range, 38–432
38–432 ng/mL),
ng/mL), whichwhich was comparable
was comparable to theatlevel
to the level 2 at
2weeks
weeks after
after immunization
immunization (Figure
(Figure 7B).7B).

Figure 7. GBSV6 induced long-lasting plasma cells and Th17 cells and provided efficient long-term
protection. Six months after the last immunization, GBSV6-specific antibodies were determined
by the ELISA, GBSV6-specific antibody-secreting B cells were determined by B cell ELISpot, and
IL-17A-secreting T cells were determined by T cell ELISpot. (A) Serum IgG, (B) vaginal IgA, (C) IgG-
and (D) IgA-secreting B cells in the bone marrow, and (E) IL-17A-secreting T cells in the spleen.
Data represent mean ± SEM (n = 10). Mice were intravaginally challenged with GBS serotype
Ib (1 × 107 ) and (F) CFUs in the vaginal lavage fluid were determined at the indicated time points.
(G) Percentages of GBS-colonized mice were calculated. Data represent the percentage of mice
colonized with GBS. ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Vaccines 2023, 11, 1459 14 of 18

Consistently, ELISpot showed that the bone marrow levels of IgA+ and IgG+ long-
lasting plasma cells were substantially increased after i.n. immunization compared to CpG
control mice (Figure 7C,D). Furthermore, long-lasting IL-17A+ T cells that responded to
GBSV6 were detected in the spleens of these immunized mice (Figure 7E). These results
indicate that i.n. GBSV6 immunization induces long-term immunity.
To assess the efficacy of the long-term immunity, mice were i.n. challenged with GBS
serotype Ib at six months after the last immunization, and CFUs in the vaginal lavage
fluid were determined. Vaginal CFUs were non-significantly lower in the immunized mice
than in control mice (Figure 7F), and by 35 days after the challenge, there were no CFUs
in the immunized mice, whereas 50% of the control mice still had CFUs (Figure 7G). This
indicates that GBSV6-induced long-lasting immunity promotes faster elimination of GBS
colonizing the vagina.

4. Discussion
GBS is a leading cause of life-threatening neonatal infections. The diversity of the
serotypes of clinical isolates necessitates GBS vaccines that confer cross-serotype protection
against the pathogen. While no licensed vaccine exists, conserved GBS proteins are being
used for GBS vaccine development for this purpose. Our results showed that i.n. GBSV6
immunization promoted GBS clearance from the vagina and maternal i.n. GBSV6 immu-
nization reduced the death of newborn mice in a serotype-independent manner. Further,
immunization induced long-term specific antibody and T cell responses and provided
long-term resistance to intravaginal challenge.
We showed that the death of newborns of immunized dams was reduced (compared to
controls) to a similar extent in both the i.m. and i.n. groups, but the increase of vaginal IgA
antibody and reduction of GBS vaginal colonization were only in the i.n. group and not the
i.m. group, indicating that local mucosal IgA significantly contributes to reduced GBS in the
vagina. GBS colonization of the reproductive tract is the primary risk factor for EOD during
delivery [47], and vaginal mucosal immunity may be critical to inhibit GBS colonization
of the reproductive tract and reduce the risk of infection for the newborn. However, we
noticed that the reduced vaginal colonization after i.n. immunization seemed not to further
reduce the death of newborns compared to i.m. immunization. The impact of the decrease
in vaginal CFUs on newborn infection needs to be explored. As GBS ascending to the uterus
from the lower genital tract of pregnant women can cause intrauterine fetal death (IUFD),
and as parenteral immunization reduces the rate of IUFD [10], it is important to ascertain
whether maternal i.n. immunization further reduces this rate. In summary, establishment of
immunity in newborns and reduction of bacterial colonization of the vagina of mothers are
two promising strategies for effectively constraining GBS diseases. Our results suggest that
i.n. GBSV6 immunization induces maternal antibodies, which establish immunity against
GBS in newborns for the prevention of newborn death. Additionally, mucosal immunity
in the reproductive tract may reduce the risk of GBS transmission from the mother to the
baby during pregnancy or at birth [48].
Mice are not a natural host of GBS and are not sensitive to vaginal infection. β-Estradiol
increases the sensitivity of mice to vaginal infection and may have some unknown effects
that affect the correctness of the interpretation of the results. Verifying the results with
different animal models is needed and will be conducted in the future.
The persistence of immunity provides long-lasting protection against reinfection. It
has been reported that mucosal immunization induces specific long-term immunity in the
genital tract and provides longer lasting protection against intravaginal challenge when
compared to systemic immunization with the same antigen. Mucosal compared to systemic
immunization is important for the induction of long-term maintenance of immunity in
the reproductive tract [49]. We demonstrated that i.n. GBSV6 immunization induced
long-lasting immunity against GBS, although the long-term serum IgG and vaginal IgA
levels were 60% and 59%, respectively, of the early levels. This long-term immunity would
maximize protection for fetus development and against infant infection [4]. Adjuvants
Vaccines 2023, 11, 1459 15 of 18

used to enhance the immunogenicity of subunit vaccines would induce potential pro-
inflammatory reactions, which have a risk of adverse reproductive, teratogenic, or fetal
developmental effects [50]. The long immunity would also allow administration of a vaccine
prior to pregnancy to reduce the possible risks of vaccination to fetus development [51].
We noticed that the mouse vaginal antibody response to GBSV6 is predominantly IgG.
Human studies have shown that in sharp contrast to other mucosal sites, in which secretory
IgA represents the dominant Ig isotype, human semen, cervicovaginal secretions, and urine
contain higher levels of IgG than IgA [52] due to the minimal associated local lymphoid
tissue in the FRT compared to other mucosal sites [53]. However, vaginal IgG, but not IgA,
significantly reduced six months after GBSV6 immunization, indicating that IgA is more
sustainable than IgG in the vagina.
GBSV6 contains most essential virulence factors of GBS and provides efficient protec-
tion. However, clinical GBS isolates in various countries or regions may carry different
combinations of virulence factors. GBSV6 can be adjusted according to the prevalence of
virulence factors in epidemic GBS strains for better targeting if necessary. Protein-based
vaccines provide convenience for the technical adjustment. On the other hand, GBSV6 can
be refined by minimizing virulence factors without affecting optimal protection.
CD4+ T helper cells (Th) play a central role in immune protection by providing help to
B cells and releasing cytokines to protect against pathogenic microorganisms [54]. Th17
cells are critical for protection against bacterial infection in mucosal sites [55]. We previously
reported that Th17 cells and the IL-17A that they release are required for efficient clearance
of group A streptococcus infections [28]. In addition, IL-17A is associated with the eventual
clearance of GBS [56]. We found that immunized mice developed a strong Th17 response
in the spleen and FRTs, suggesting that the Th17 response plays an important role in
clearing GBS.
The presence of non-circulating, tissue-resident memory T cells (Trms) in the desired
tissues is important for long-term vaccine-induced immunity in these tissues, which reduces
the incidence of infection [57,58]. However, we did not find these cells in the vaginal tissue,
though GBSV6-specific memory T cells were detected in the spleen. Evidence indicates that
Trms preferentially populate the site of immunization [59,60]. Thus, efficient generation of
Trms in the FRT requires intravaginal stimulation. Whether combined i.n. and intravaginal
immunization can strengthen the long-lasting protective immunity in the vagina will be
tested in our future study.

5. Conclusions
In this study, we demonstrated that immunization with a set of six conserved virulence
factors involved in GBS pathogenesis through the i.n. route induces serotype-independent
immunity both systemically and in the vagina, and thus provides protection against GBS.
The immunity is long-lasting and reduces the death of newborn mice and GBS colonization
of the vagina. The results suggest that multiple conserved virulence factors and the i.n.
route can be considered as strategies for advancing GBS vaccine development.

Supplementary Materials: The following supporting information can be downloaded at: https://www.
mdpi.com/article/10.3390/vaccines11091459/s1, Figure S1: Purified recombinant proteins of GBSV6.
Purified recombinant proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and stained with 0.25% Coomassie brilliant blue. Lane 1, molecular weight marker
(MW); lane 2, SrtA; lane 3, ScpB; lane 4, CspA; lane 5, Srr2; lane 6, BibA; lane 7, FbsB. Table S1: Primers
Used for Cloning GBSV6 genes. Underlined nucleotides denote enzyme restriction sites.
Author Contributions: Funding acquisition, B.W.; experiment design, B.W. and J.W.; investigation,
J.W., W.L. and N.L.; methodology, J.W. and W.L.; software, N.L.; writing—review and editing, J.W.
and B.W. All authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by Beijing Varnotech Biopharm Ltd. to B.W.
Vaccines 2023, 11, 1459 16 of 18

Institutional Review Board Statement: The study was conducted according to the guidelines of
the Declaration of Helsinki. All animal-related experimental protocols applied in this study were
conducted under the standards of the Committee on the Ethics of Animal Experiments of the Institute
of Microbiology, Chinese Academy of Sciences (SQIMCAS20211128).
Informed Consent Statement: The collection and use of human serum samples were approved by
the Ethics Committee of Beijing Children’s Hospital. Informed consent was obtained from all subjects
prior to enrollment in the study.
Data Availability Statement: All data that this study is based upon are available from the corre-
sponding author upon request.
Acknowledgments: We are very grateful for the financial and technical support provided by
Varnotech Biopharm Ltd. J.W. and W.L. are current employees of Varnotech Biopharm Ltd. W.L.
reports personal fees and grants from VARNO during the conduction of the study. J.W. and B.W.
report grants from Varnotech Biopharm Ltd. during the conduction of the study.
Conflicts of Interest: All authors declare no conflict of interest.

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