Pregnancy Vaccination with Gold Glyco-Nanoparticles Carrying Listeria monocytogenes Peptides Protects against Listeriosis and Brain- and Cutaneous-Associated Morbidities

Listeriosis is a fatal infection for fetuses and newborns with two clinical main morbidities in the neonatal period, meningitis and diffused cutaneous lesions. In this study, we vaccinated pregnant females with two gold glyconanoparticles (GNP) loaded with two peptides, listeriolysin peptide 91–99 (LLO91–99) or glyceraldehyde-3-phosphate dehydrogenase 1–22 peptide (GAPDH1–22). Neonates born to vaccinated mothers were free of bacteria and healthy, while non-vaccinated mice presented clear brain affections and cutaneous diminishment of melanocytes. Therefore, these nanoparticle vaccines are effective measures to offer pregnant mothers at high risk of listeriosis interesting therapies that cross the placenta.


Introduction
Human listeriosis is a food-borne invasive illness caused by the pathogen Listeria monocytogenes (LM) that causes infections in pregnant women, infants, the elderly, and the immune-compromised.While infections are rare among the healthy population, listeriosis is one of the most lethal bacterial diseases for fetuses and newborns [1][2][3][4][5][6][7].Nevertheless, pregnant women who get infected experience only mild symptoms, making the diagnosis very difficult, even if fetuses are fatally infected [1].Therefore, vaccination of pregnant women appears as the most cost-effective measure to deal with this deadly pathogen in pregnancy, as it may be responsible for a high number of non-diagnosed spontaneous abortions.
Our group has been preparing vaccines for this pathogen in different formats.We have successfully developed dendritic cell vaccines loaded with LM peptides and deciphered that two peptides from this pathogen's virulence factors, listeriolysin O (LLO) and glyceraldehyde-3phosphate-dehydrogenase (GAPDH), LLO 91-99 and GAPDH 1-22 , conferred significant listeriosis protection in two mice models sensitive and resistant to listeriosis [8][9][10].We have also prepared a gold glyconanoparticle (GNP) coupled to the LLO 91-99 peptide formulated with an adjuvant and achieved good vaccine effectiveness in adult mice [11].Indeed, the application of nanotechnology in immunology is creating big expectations in the field of vaccination [12] and many examples of nanoparticles (NP) have appeared in the literature [13,14].Nanoparticle-based vaccine candidates have been developed in order to improve the adjuvant effect of the vaccine formulations [15,16], to deliver immunologically active components to target sites [17] and to trigger immune-modulation of inflammatory responses depending on the mechanisms of NP uptake and interactions with immune cells [18].Among synthetic NPs, GNPs have recently been reported to be able to potentiate the adaptive immune response towards carbohydrate antigens based on the concept of multivalency and depending on the antigen type and loading [19][20][21].In fact, nanoparticles are promising vector systems to explore vaccination during pregnancy against infectious agents [22].The interesting results obtained with GNP-LLO 91-99 [11] prompted us to prepare other glyconanoparticles, GNP-GAPDH 1-22 , and examine the ability of these two vaccine vectors, GNP-LLO 91-99 and GNP-GAPDH 1-22 , to protect fetuses from listeriosis.For this purpose, we vaccinated pregnant mothers with the above-mentioned glyconanoparticles formulated with adjuvants and followed LM infection during pregnancy and the neonatal period as well as the morbidities associated with neonatal listeriosis.

Nanoparticle Vaccine Effectiveness in Pregnancy Amilorates Listeriosis-Associated Morbidities
Neonatal listeriosis is characterized by at least three morbidities in newborns: stillbirths, central nervous sytem affection and cutaneous diffused lesions [23,24].We vaccinated pregnant mothers of C57BL/6 mice (n = 2) at day 9 of gestation (E9) with two prepared nanoparticle vaccines, GNP-LLO 91-99 and GNP-GAPDH 1-22 , formulated with Advax TM adjuvant, followed by three days of challenge with LM WT on day 16 of gestation (E16).We prepared three groups of pregnant mothers (n = 2), vaccinated as above, non-vaccinated but challenged with LM WT (NV) and non-vaccinanted and non-infected (control).Control mothers non-vaccinated and non-infected gave birth to nine pups (lower left P0 images in Figure 1), a normal number in C56BL/6 mice that usually deliver six to nine pups.Similarly, GNP-GAPDH 1-22 -or GNP-LLO 91-99 -vaccinated and LM WT -challenged mothers delivered nomal numbers of pups, nine and eight, respectively (middle left P0 images in Figure 1).However, NV-and LM WT -challenged mothers only delivered two to three pups (upper P0 images in Figure 1).The reduction of neonates in these groups of NV mothers was due to stillbirths or resorbed fetuses as we detected in the mother's uterus (upper left P0 image in Figure 1).On day 4 after birth (P4), all pups were explored for clinical data, weight, length, coordination movement tests and general and skin observations with a magnifying lens.Clinical data (Table 1) were normal in the control group and GNP-GAPDH 1-22 -and GNP-LLO 91-99 -vaccinated mothers, but they were clearly impaired in the group of NV mothers.The NV group of pups showed weight loss, no ability to move in the metric paper test and a light grey color and wrinkled skin compared to the black and normal skins of vaccinated and control groups (Table 1 and NV images under P4 in Figure 1).re 1. P0 corresponds to day 1 and P4 to day 4 of pups born to pregnant mothers.Group nant mothers were the following: non-vaccinated and challenged with Listeria monocytoge WT ) (NV), vaccinated and challenged with LM WT (gold glyconanoparticles listerioly tide 91-99 (GNP-LLO91-99)or gold glyconanoparticles glyceraldehyde-3-phosph ydrogenase 1-22 peptide (GNP-GAPDH1-22)) or non-vaccinated and non-infected with LM trol).P4-brain corresponds to brains of P4 neonates.These results suggested that pups born to NV mothers showed central nervous system retardation and enlarged heads and cutaneous immaturation.To confirm these morbidites we sacrificed P4 neonates from the four groups of pregnant mothers.Brains were collected and examined (P4-brain images in Figure 1) and we detected softer cranial covers in pups born to NV mothers (NV images under P4-brain in Figure 1), a significant reduction of blood vessels confirmed by immunohistochemical analysis and a reduced number of melanocytes in the skin with strong apoptotic staining (Table 2).
a Relative melanocytes, correspond to the number of melanocytes per 1 mm of skin measured by immunohistochemistry; b Apototic melanocytes, correspond to the percentages of melanocytes positive for the TUNNEL staining; c Blood vessels were observed in whole brains with magnifying lens and classified as few or normal.

Nanoparticles Vaccine Reduced the Number of Viable Bacteria in Microglia and Pro-Inflammatory Cytokine Production
We observed that P4 pups born to NV mothers infected with fluorescent green fluorescence protein Listeria monocytogenes (GFP-LM WT ) presented high microglía (MG) infection (green fluorescence in P4-brain, in panel a, Figure 2).MG were characterized by the macrophage F4/80 marker (red fluorescence in P4-brain, panel a, Figure 2).However, we did not observed any bacteria in other brain cells such as neurons as detected with the anti-a-tubuline antibody (blue fluoresnce in P4-brain, panel a, Figure 2), similar to results of in vitro infection of mixed MG cultures [25].Moreover, purified and isolated MG of P4 pups born to NV mothers presented a severe infection using GFP-LM WT (green fluorescence in P4-MG images, panel a, Figure 2).Numbers of colony-forming-units (CFU) in P4 ewborn pups (NB) born to NV mothers confirmed a high number of viable bacteria, with 6.2 × 10 2 CFU/mL detected in MG.We detected very few bacteria in MG of P4 pups born to GNP-LLO 91-99 -and GNP-GADPH 1-22 -vaccinated mothers, with 6.2 and 2.2 CFU/mL, respectively (panel b, Figure 2).Next, we also explored listeriosis in the mothers and observed no viable CFU in livers or spleens from GNP-LLO 91-99 -or GNP-GAPDH 1-22 -vaccinated mothers (data not shown).However, we detected 2.40 × 10 5 CFU/mL in livers of NV mothers that corresponded with 25-fold growth of bacteria compared to initial inoculation with 10 4 CFU/mice (panel b, Figure 2) and suggested a normal listeriosis infection in these NV mothers.The low CFU in spleens of NV mothers, 6.5 × 10 CFU/mL, indicated these pregnant mothers cleared the infection, while placental transmission was fatal for their fetuses, and after four to six days all pups died (data not shown).

Nanoparticles Vaccine Reduced the Number of Viable Bacteria in Microglia and Pro-Inflammatory Cytokine Production
We observed that P4 pups born to NV mothers infected with fluorescent green fluorescence protein Listeria monocytogenes (GFP-LM WT ) presented high microglía (MG) infection (green fluorescence in P4-brain, in panel a, Figure 2).MG were characterized by the macrophage F4/80 marker (red fluorescence in P4-brain, panel a, Figure 2).However, we did not observed any bacteria in other brain cells such as neurons as detected with the anti-a-tubuline antibody (blue fluoresnce in P4-brain, panel a, Figure 2), similar to results of in vitro infection of mixed MG cultures [25].Moreover, purified and isolated MG of P4 pups born to NV mothers presented a severe infection using GFP-LM WT (green fluorescence in P4-MG images, panel a, Figure 2).Numbers of colonyforming-units (CFU) in P4 ewborn pups (NB) born to NV mothers confirmed a high number of viable bacteria, with 6.2 × 10 2 CFU/mL detected in MG.We detected very few bacteria in MG of P4 pups born to GNP-LLO91-99-and GNP-GADPH1-22-vaccinated mothers, with 6.2 and 2.2 CFU/mL, respectively (panel b, Figure 2).Next, we also explored listeriosis in the mothers and observed no viable CFU in livers or spleens from GNP-LLO91-99-or GNP-GAPDH1-22-vaccinated mothers (data not shown).However, we detected 2.40 × 10 5 CFU/mL in livers of NV mothers that corresponded with 25-fold growth of bacteria compared to initial inoculation with 10 4 CFU/mice (panel b, Figure 2) and suggested a normal listeriosis infection in these NV mothers.The low CFU in spleens of NV mothers, 6.5 × 10 CFU/mL, indicated these pregnant mothers cleared the infection, while placental transmission was fatal for their fetuses, and after four to six days all pups died (data not shown).

Discussion
The objective of this study was to identify an optimal vaccine formulation to protect against neonatal listeriosis.Preconceptual vaccination with attenuated L. monocytogenes strains does not protect against fetal wastage or placental-fetal invasion [30], prior to or during pregnancy.Therefore, different vaccination vectors or systems should be used to protect against infections of vertical transmission.In this regard, dendritic vaccines emerged as effective vectors able to confer significant protection against systemic listeriosis in different mice models, either sensitive or more resistant to LM infection [8,9,23].Further, dendritic vaccines loaded with peptides from virulence factors of LM such as lysteriolysin O (LLO) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were effective for protection, but they are expensive measures to offer all pregnant women at risk of listeriosis.In this regard, nanoparticles are promising vaccine vectors with interesting properties such as safety, biocompatibility, and versatility to load with different ligands and directionality to dendritic cells with chemical modifications such as carbohydrates [31][32][33].
We previously reported that gold glyconanoparticles loaded with a listeriolysin O peptide, GNP-LLO 91-99 , when formulated with adjuvants such as Advax TM , conferred significant protection against systemic listeriosis in adult animal models [11,23].Taking advantage of this nanoparticle system, we also designed a similar vector coupled with another peptide of the GAPDH virulence factor, GAPDH 1-22 , which showed promising properties in dendritic cell vaccines [8,9].Here, we presented that both GNP-LLO 91-99 and GNP-GAPDH 1-22 , nanovaccines formulated with Advax T004D conferred good protection during pregnancy against neonatal listeriosis.GNP-GAPDH 1-22 nanovaccines appeared more effective than GNP-LLO 91-99 nanovaccines as they showed lower CFU numbers in MG.Moreover, these nanovaccines reduced central nervous system (CNS) and skin-associated morbidities as well as pregnancy complications.
Invasive listeriosis in pregnancy is a fatal infection for the fetuses, causing complications such as spontaneous abortions stillbirths in 20% of the cases, especially if infection occurs early in the pregnancy; preterm delivery and neonatal infection are also observed [1,34].70% of surviving neonates born to mothers infected with LM, presented trans-placental transmission of bacteria.Neonatal listeriosis manifestations imply meningitis, brain abscesses, diffused skin lesions and rash, fever and lethargy [1][2][3][4][5][6][7]23,26].While not exactly similar, neonatal experimental listeriosis inoculating pregnant mice with pathogenic LM reproduces most of the clinical symptoms [26][27][28]34].In this regard, our results here detecting resorbed fetuses in mice mothers might reflect human stillbirths; the 2-to 3-fold reduction in neonates at birth might mimic spontaneous abortions, all clinical complications related to pregnancy [1].We also detected CNS-associated morbidities such as enlarged heads, softer cranial covers and a lack of coordinated movement that resembled CNS impairment [24].Finally, the lower number of skin melanocytes due to apoptosis we detected in P4 pups born to NV mothers appeared comparable to cutaneous diffused lesions of granulomatosis infantiseptica forms of severe listeriosis in neonates [23].All these above-mentioned listeriosis-associated morbidities disappeared in pregnant mothers pre-vaccinated with GNP-GAPDH 1-22 and GNP-LLO 91-99 nanovaccines and in neonates born to these vaccinated mothers.Therefore, they highlighted the ability of these nanovaccines to cross the plancental barrier and protect the fetuses.
Moreover, the ability of these nanovaccines to shift the cytokine pattern of MG [35] towards a Th1-IL-12-dependent pattern is supposed a good benefit since IL-12 production seems associated with vaccine efficiency and long-term protection [8,11,28], but also because acute Th2 cytokines such as IL-6 [29] were significantly reduced after vaccination with GNP-GAPDH 1-22 and GNP-LLO 91-99 nanovaccines.
We concluded that GNP-LLO 91-99 and GNP-GAPDH 1-22 nanovaccines are effective measures for vaccination against listeriosis during pregnancy, with GNP-GAPDH 1-22 being more potent than GNP-LLO 91-99 nanovaccines.The disappearance of listeriosis morbidities is an added value of these nanovaccines that revealed their powerful action.
Animals.We used C57BL/6 mice from our animal facilities at the University of Cantabria at 8-12 weeks old.Three female and one male were mated and assessed for the appearance of vaginal plug denoting first embryonic day of pregnancy.
Pre-natal vaccination.Pregnant C57BL/6 female mice were vaccinated or not (NV) at day 9 of gestation (E9) with intravenously (i.v) via the lateral tail vain with GNP-LLO 91-99 or GNP-GAPDH 1-22 formulated with Advax TM (5 µg of nanoparticles and 250 µg of adjuvants/mice).Next, at 16 days of gestation (E16) all mice, vaccinated and NV were inoculated i.v with 100 µL of a LM WT bacterial suspension in saline (1 × 10 5 CFU/mL) (LM WT infected mothers).We also preserved a group of mothers NV and inoculated with 100 µL of saline (Control).All animals were daily examined.At E20 we detected two to three pups born to LM WT infected mothers and NV, while six to eight pups born to control and vaccinated mice.Four days after birth, P4 postnatal pups born to LM WT infected and vaccinated (GNP-LLO 91-99 or GNP-GAPDH 1-22 ) or NV mothers were sacrificed to obtain cerebellum for preparation of mixed microglia and subsequent isolation of primary microglial cultures for CFU quantification and cytokine quantification.LM WT infected and vaccinated, NV and control mothers were bled for cytokine analysis and sacrificed to obtain uterus for observation of resorbed fetuses and spleens and livers for CFU quantification.
Clinical tests.Clinical test were performed on all pups born to mothers vaccinated, NV or control on days P0 and P4.Test include weight, length, coordination-movement assays placing pups in a metric paper and recording the centimeters of movement after 5 min, general and cutaneous screening for lesions and wounds with a magnifying glass.
Microglia isolation.Microglial cultures have been described [37,38] and detailed procedures for obtaining mixed microglial cultures and purified primary microglia were reported [24].In brief, mixed microglial cell cultures were obtained from cerebellum at P4 until complete neuronal differentiation for seven days as described [37].Microglial cultures shaked at 200 times/min for 30 min and cells in supernatants re-plated in 24-well plates.MG cells were 90% double positive CD11b + CD45 + and 80% double positive for F4/80 + IA b+ as measured by flow cytometry (FACS).Supernatants of MG cells were filtered and stored at −80 • C for cytokine analysis by FACS and next, MG cells were lysed and plated in BHI agar plates for CFU analysis.
Confocal assays.Cells used for confocal microscopy were fixed in 3% paraformaldehyde.Fluorescence labeling and confocal microscopy were performed as previously described [24].

able 1 .
Clinical data of pups (P4) born to vaccinated, non-vaccinated (NV) or control mothers

Figure 2 .
Figure 2. (a) P4-brain image corresponds to mixed microglía (MG) cultures of cerebellum brains showing green fluorescence of green fluorescence protein Listeria monocytogenes (GFP-LM), red fluorescence of the macrophage marker F4/80 and blue fluoresecence of neuron tubuline.P4-MG corresponds to purified MG of mixed cultures showing green fluorescence of GFP-LM, red fluorescence of actin cytoskeleton stained with TRITC-phalloidin and blue fluorescence of tubuline; (b) colony-forming-units (CFU)/mL in MG of newborn P4 pups (NB) born to NV mothers, GNP-LLO 91-99 or GNP-GAPDH 1-22 vaccinated mothers.CFU/mL in livers and spleens of NV mothers are also examined; (c) Cytokines levels (pg/mL) in supernatants of MG of newborn P4 pups born to mothers as in Figure 1.

Table 1 .
Clinical data of pups (P4) born to vaccinated, non-vaccinated (NV) or control mothers.
a Four groups of pregnant mothers were prepared: non-vaccinated (NV), GNP-LLO 91-99 -vaccinated, GNP-GAPDH 1-22 -vaccinated and controls (non-vaccinated).NV and vaccinated groups were challenged with Listeria monocytogenes (LM WT ) but not the control group.All P4 pups were examined for clinical data; b Coordination movement test measures in a 10 cm metric paper the length (cm) achieved by each group of mice in 5 min.Results are the mean ±SD of triplicates; c Cutaneous test with magnifying lens to confirm skin color (black, grey or white) and aspect (normal or wrinkled).

Table 2 .
Number of relative melanocytes and blood vessels.

Table 2 .
Number of relative melanocytes and blood vessels.Relative melanocytes, correspond to the number of melanocytes per 1 mm of skin measured by immunohistochemistry; b Apototic melanocytes, correspond to the percentages of melanocytes positive for the TUNNEL staining; c Blood vessels were observed in whole brains with magnifying lens and classified as few or normal. a

Table 3 .
Cytokine levels of purified microglía (MG) from P4 neonates born to vaccinated or NV mothers.

IL6 (pg/mL) a IL-12 (pg/mL)
a Cytokines are measured by flow cytometry and results expressed as concentration of pg/mL ± SD of triplicates.