Inflammatory and Immune Responses during SARS-CoV-2 Infection in Vaccinated and Non-Vaccinated Pregnant Women and Their Newborns

Background. Pregnant women are more susceptible to severe disease associated with SARS-CoV-2 infection. We performed a prospective study to analyze the inflammatory and immune profile after SARS-CoV-2 infection occurring in vaccinated or non-vaccinated pregnant women and their newborns. Methods. Twenty-five pregnant women with SARS-CoV-2 infection were enrolled, and sixteen cord blood samples were obtained at delivery. Results. We observed that IL-1β, TNF-α, Eotaxin, MIB-1β, VEGF, IL-15, IL-2, IL-5, IL-9, IL-10 and IL-1ra levels were significantly higher in vaccinated than non-vaccinated mothers. Furthermore, the newborns of the vaccinated mothers produced higher levels of IL-7, IL-5 and IL-12 compared to the newborns of non-vaccinated mothers. Anti-Spike (S) IgG levels were significantly higher in all vaccinated mothers and their newborns compared to the non-vaccinated group. We found that 87.5% of vaccinated women and 66.6% of non-vaccinated women mounted an S-specific T-cell response quantified by ELISpot assay. Moreover, 75.0% of vaccinated mothers and 38.4% of non-vaccinated mothers showed S-specific CD4+ T-cell proliferative response. The T-helper subset response was restricted to CD4+ Th1 in both vaccinated and non-vaccinated women. Conclusion. A higher level of cytokines, IgG antibodies and memory T cells was noted in the vaccinated women. Furthermore, the maternal IgG antibody trans-placental transfer occurred more frequently in vaccinated mothers and may protect the newborn.


Introduction
Pregnancy is a state of altered immunity, causing pregnant women to be more vulnerable to viral infections, including SARS-CoV-2 infection. Data from previous pandemics, such as those caused by SARS, influenza virus H1N1 [1] and MERS [2], show that pregnant women are more susceptible to serious illness with adverse outcomes and display greater mortality rates than the general population. However, a recent meta-analysis demonstrates that pregnant women with COVID-19 have similar clinical characteristics and outcomes as the non-pregnant population [3]. Despite the limited evidence of vertical transmission [3], infected women are at significantly higher risk for cesarean delivery, preterm birth and adverse neonatal outcomes [4][5][6][7] compared to the general pregnant population. Moreover, increased levels of pro-inflammatory cytokines, IL-8, IL-10 and IL-15, have been observed in the circulation of pregnant women with SARS-CoV-2 infection and their neonates, even in the absence of placental infection [8]. Gee and colleagues showed an increase in cytokines in cord plasma following the presence of an inflammatory profile in the mothers but also altered immune cell functionality in neonates exposed to SARS-CoV-2 at any point during gestation [9]. Notwithstanding their higher risk, pregnant and lactating women were not included in any initial COVID-19 vaccine trials, although the first vaccine trial with pregnant women began in February 2021 (Pfizer/BioNTech, ClinicalTrials.gov identifier: NCT04754594). The preliminary findings did not show additional complications among pregnant women who received BNT162b2 (Pfizer/BioNTech) and mRNA-1273 (Moderna) compared to non-pregnant persons [10][11][12][13]. Moreover, the proportions of adverse pregnancy and neonatal outcomes (i.e., preterm birth, congenital anomalies and neonatal death) among mothers with completed pregnancies appeared to be similar to the published incidences in pregnant populations studied before the COVID-19 pandemic [14][15][16].
Numerous studies have reported the effects on the fetus following vaccination in pregnancy [17,18]. The maternal antibodies increased by the vaccination during pregnancy cross the placenta and are transferred in the umbilical cord blood at birth [19,20], remaining detectable in the blood of more than half of newborns at 6 months [21]. In addition, many studies indicate that anti-SARS-CoV-2 IgG and IgA antibodies were transmitted to newborns through vaccinated mother's milk, resulting in a probable protective role [22,23]. In this prospective study, we evaluated the inflammatory and immune profile in pregnant women vaccinated and non-vaccinated during SARS-CoV-2 infection.

Study Design
A prospective observational study was conducted to evaluate the cytokine, antibody and T-cell responses in vaccinated and non-vaccinated SARS-CoV-2-infected pregnant women and their newborns. The study included 25 pregnant women (median age 32 years; range  enrolled between January 2022 and February 2022 at the Obstetrics and Gynecology Clinics of Fondazione IRCCS Policlinico San Matteo (Table 1). All pregnant women were hospitalized to treat preterm labor, hypertension preeclampsia and cholestasis or because they were close to delivery (median gestational weeks: 39.0; range 16.0-42.0). of enrollment, all of them were diagnosed with SARS-CoV-2 infection via RT-PCR by taking a nasal swab from each of them. Blood and serum samples were obtained from all women subsequently. In addition, all information on the SARS-CoV-2 vaccine administered to the patients was collected.
Blood and serum samples were obtained from all women at time of enrollment, after diagnosis of SARS-CoV-2 infection via nasal swab testing (median days since diagnosis of infection: 2; range 0-20). Cord blood samples were collected at delivery from 17 newborns from 17 mothers (n = 6 from vaccinated mothers; n = 11 from non-vaccinated mothers). The study was approved by the local Ethics Committee (P-20200046007), and all subjects gave written informed consent.

Quantification of Cytokines
Serum concentrations of cytokines, chemokines and growth factors were measured in duplicate using BioPlex Pro Human Cytokine Screening Panel (27-Plex #M500KCAF0Y, Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions. Data were obtained with BIO-PLEX manager software 6.0.

Antibody Response
Serum samples from mothers and newborns were tested for SARS-CoV-2 Anti-Spike (S) and anti-Nucleocapsid (NCP) IgG antibodies using ELISA (Euroimmun, Lübeck, Germany), according to the manufacturer's instructions. The semi-quantitative results were expressed as a ratio (RU/mL) with respect to an internal calibrator: a ratio of <0.8 was considered negative, ≥1.1 was considered positive and intermediate results were considered borderline.

T-Cell Response (IFNγ Production)
ELISpot assay was used to evaluate Spike-specific T-cell response (IFNγ production), according to the following protocol (Cassaniti et al., CMI 2021). Briefly, peripheral blood mononuclear cells (PBMCs) at a concentration of 2 × 10 5 /100 µL culture medium per well were stimulated for 24 h in 96-well plates (coated with anti-IFN-γ monoclonal capture antibody) with peptide pools (15 mers, overlapping by 10 amino acids, Pepscan, Lelystad The Netherlands) representative of the Spike protein (S), at a final concentration of 0.25 µg/mL. Phytoheamagglutinin (PHA; 5 µg/mL) was used as positive control and medium alone as negative control. Responses ≥ 10 net spot forming cells (SFCs)/million PBMCs were considered positive based on background results obtained with negative control (mean SFC + 2SD).

Statistical Analysis
Descriptive statistics for quantitative data was reported as median and range. Comparison between two groups was performed using the Mann-Whitney U-test, and correlation was calculated with Spearman's method. All tests were two-tailed, and p value < 0.05 was considered statistically significant. GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA, USA) was used for analyses. To draw the heatmap, we used GraphPad Prism 8.0 and Python 3.8.5 in the Jupyter notebook environment. In this case, we used "pandas" and "seaborn" modules. To calculate the factor analysis, we used Python 3.8.5. FactorAnalyzer. FactorAnalyzer was applied to statistically analyze the data in the Jupyter notebook. Factors were selected based on Eigenvalues greater than 1. Cytokines that were more than 50% correlated with each factor were classified as highly correlated.

Characteristics of the Pregnant Women Analyzed
As reported in Table 1, no difference in age, race, co-morbidities, clinical parameters, symptoms and therapy between vaccinated and non-vaccinated women was observed. Four women (16%) had a symptomatic infection and two of them, showing fever and cough associated with rhinitis or pharyngitis, were treated with azitromicin and steroid (betamethasone). The remaining 21/25 women (84%) had no symptoms. Of all 25 patients, 2 (8.0%) had a previous SARS-CoV-2 infection eight and sixteen months before enrollment. All the newborns (NBs) were negative for SARS-CoV-2 RNA in nasal swabs collected after birth and without neonatal complications.

Cytokine Production
The serum of vaccinated/non-vaccinated mothers and their NBs was tested to evaluate the concentration and correlation of 27 different cytokines, chemokine and growth factors. In the vaccinated women, the network of cytokines was intact. Only one chemokine, RANTES (CCL5), showed a lower correlation with other cytokines, except for IP-10 ( Figure S1A). In the non-vaccinated women, there was no correlation between some of the important chemokines such as IP-10 (CXCL10) with other important chemokines, cytokines and growth factors ( Figure S1B). The virus even dismantles the cytokine network with disabling growth factors, such as IL-7 and PDGF-bb. The infection manipulates the cytokine network by affecting T-cell cytokines, IL-13 and correlation with other key cytokines and chemokines. In addition, the cytokine serum concentration was higher in vaccinated than non-vaccinated mothers but not symptomatically ( Figure 1). In particular, IL-1β, TNF-α, Eotaxin, MIP-1β, VEGF, IL-15, IL-2, IL-5, IL-9, IL-10 and IL-1ra levels were significantly higher in vaccinated than non-vaccinated mothers. Moreover, the NBs of the vaccinated mothers produced significantly (or close to significance) higher levels of IL-7, IL-5 and IL-12 compared to the NBs of non-vaccinated mothers (Figure 2A,B). No significant difference for the other cytokines was observed. A correlation in the concentration of TNF-α, IL-6, IL-17, MIP-1α and G-CSF between non-vaccinated mothers and their NBs was noted, as reported in Table 2, but no correlation in the group of vaccinated mothers and their NBs was observed.     In the second factor, IL-7 and PDGF-bb/PDGFB were highly correlated, while in the third and fourth factors, MIP-1β/CCL3 and IL-13 were highly regulated, respectively, Table S1. The vaccinated pregnant women were categorized into five factors based on Eigenvalues, IL-13, eotaxin (CCL11, CCL24, CCL26), FGF, G-CSF, GM-CSF, IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-15, IL-17, MIP-1α/CCL4, MIP-1β/CCL3, RANTES/CCL5, TNF-γ and VEGF were highly correlated. While in the second factor, IL-8, IL-17, MCP-1/CCL2 and IL-9 were correlated. In the third factor, IP-10 and RANTES/CCL5 were correlated. The fourth factor was highly correlated with PDGF-bb/PDGFB and IL-12. Finally, the fifth factor was correlated with IL-13, Table S2.

Antibody Response
All vaccinated mothers and their newborns showed detectable levels of anti-S IgG (median ratio: 6.4, range 2.4-7.9 in the mothers, and 6.5, range 3.0-7.6 in the NBs, respectively), which were higher compared to non-vaccinated mothers and their NBs (median ratio: 0.55, range 0.1-6.9 and: 0.2, range 0.2-7.9, respectively), among whom only 4/16 (25%) women and 3/11 (27%) NBs showed anti-S IgG. Two women had a previous SARS-CoV-2 infection before the current pregnancy, but only one (vaccinated) showed anti-S and -NCP IgG antibodies. Therefore, the anti-S IgG levels of vaccinated mothers and their NBs were significantly higher than the levels of non-vaccinated mothers (p = 0.0002) and their NBs (p = 0.0108) ( Figure 3A). There was no significant difference between anti-NCP IgG levels in vaccinated mothers compared to antibody levels in non-vaccinated mothers and between the NBs of both groups (p = 0.396 and p = 0.532, respectively; Figure 3B).
Among T-helper subsets, the response was restricted to CD4 + T h 1 in both vaccinated and non-vaccinated women (Supplementary Figure S2A). Instead, no S-specific CD4 + T-cell proliferative response was observed in the T h 2 and T h 17 subsets (Supplementary Figure S2B,C and Table S3).

Discussion
In this study, we evaluated: (i) cytokine profile, (ii) anti-S and anti-NCP IgG antibody levels and (iii) S-specific T-cell response in a cohort of vaccinated and non-vaccinated pregnant women experiencing SARS-CoV-2 infection.
We reported different levels of pro-inflammatory or anti-inflammatory cytokines, chemokines, growth factors and T-helper (T h ) cytokines between vaccinated and nonvaccinated mothers and their NBs. According to Figure S1, the high correlation of cytokines decreases the severity of COVID-19 in vaccinated mothers. In addition, the RANTES rise was in parallel with an increase in the secretion of IL-10 and IL-1α [24].
Interestingly, our results, in parallel with Buszko and colleagues [25], demonstrated that in SARS-CoV-2-infected patients, there is no correlation between IP-10 and IFN-γ. Escalation of the IP-10 is correlated with the severity of disease symptoms and the persistence of COVID-19 after infection. COVID-19 even impacts the cytokine network by accelerating the production of the cytokines, such as PDGF-bb and IL-7.
Bronchial fluids contain PDGF in COVID-19 patients [26]. The role of the PDGF is to recruit thrombocytes and mast cells to the upper part of the respiratory system [27]. In addition, IL-7 plays a critical role in refreshing the exhausted T cells in the immune responses. IL-7 affects IFN-γ production and T-cell proliferation during SARS-CoV-2 infection. IL-13 orchestrates the M2 macrophages, eosinophils, mucosal production by epithelial cells, fibrosis and metaplasia of the cells in the lower and upper part of the respiratory system [28]. In patients with severe symptoms, COVID-19 manipulates the immune cells to produce a higher amount of IL-13. The heatmap in Figure S1B demonstrates a lack of correlations and an interrupted correlation between IL-13 and the other main cytokines. However, since symptomatic women were limited in number, we could not analyze the potential associations between cytokine profile and severity of infections.
In addition, the serum concentration of the cytokines belonging to the different classes analyzed was higher in vaccinated mothers compared to non-vaccinated mothers. This phenomenon is likely to be a consequence of a prompter response to SARS-CoV-2 infection in vaccinated women. Conversely, only higher levels of IL-7, IL-5 and IL-12 were noted in the NBs of vaccinated mothers than in those of non-vaccinated mothers. The elevated levels of cord serum cytokines are probably derived directly from the newborns in response to SARS-CoV-2 maternal infection rather than from maternal transfer [9]. These may explain the different cytokine profiles observed in NBs and their mothers.
In a recent study on SARS-CoV-2 infection during pregnancy, the maternal plasma cytokine analysis revealed significantly elevated IP-10 and IL-1β levels in mothers with recent or ongoing SARS-CoV-2 infection compared to mothers who recovered from previous infection, while IL-10, CXCL8 and IL-6 levels were similar between the two groups. Moreover, neonates born to mothers with recent or ongoing infection express higher plasma levels of IL-10 and CXCL8 than their paired mothers [9].
However, we did not observe a clear difference in the cytokine profile in NBs to vaccinated or non-vaccinated mothers with SARS-CoV-2 infection, as instead observed in maternal serum. The vaccinated mothers presented a constructed network of cytokines compared to non-vaccinated mothers, resulting from an already existing immunological response.
The higher levels of cytokines observed in our group of vaccinated women experiencing SARS-CoV-2 infection during pregnancy are in agreement with recent studies that associated high cytokine production with improved antibody responses to SARS-CoV-2 infection occurring after the administration of the vaccine [36,37]. In particular, in addition to pro-inflammatory cytokines, we also observed a higher level of anti-inflammatory cytokines (IL-10 and IL-1ra), which may contribute to counteracting and containing the inflammatory response to the infection.
Moreover, based on the results, we analyzed each factor closely for its biological process. Utilizing the "ENRICHER" system biology (https://maayanlab.cloud/Enrichr/, accessed on 1 January 2023) helped us to analyze the biological process behind the different factors. In non-vaccinated women, the first factor was mostly taking part in eosinophil migration and chemotaxis. The second factor has a key role in glomerular mesangial cell proliferation. The cells play key roles in IL-1 and PDGF production [38]. The third-factor main biological process is positively regulating the NK cells' chemotaxis. In addition, the fourth factor is involved in the regulation of complement-dependent cytotoxicity. In the vaccinated pregnant women, the first factor has the same function as the first factor in non-vaccinated participants, eosinophil migration and chemotaxis. The second factor has a key role in inflammatory responses. The third factor positively regulates the NK cells' chemotaxis process. The fourth factor affects glomerular mesangial cell proliferation. The fifth factor regulates complement-dependent cytotoxicity.
Based on factor analysis in the vaccinated pregnant women, the second factor that takes part in inflammatory responses is different from non-vaccinated women. Due to angiotensin-converting enzyme 2 (ACE2) augmentation in pregnant women's lungs, they show fewer symptoms after SARS-CoV-2 infection. Increasing the frequency of ACE2 in the lungs of pregnant women reduces the inflammatory responses in their lungs [39]. In addition, the number of plasmacytoid dendritic cells (pDCs) is decreased in pregnant women. pDCs are critically important for inflammatory responses [40]. Furthermore, the phagocytosis phenomenon of neutrophils and monocytes is decreased in pregnant women. The consequence of phagocytosis reduction is a lower production of cytokines that take part in inflammatory responses. Moreover, steroidal hormones, such as estradiol (E2) and progesterone (P4), suppress the inflammatory responses in pregnant women [41]. Thus, reduced inflammatory responses in pregnant women favor the virus in a tug of war with immune responses. As our factor analysis demonstrates, a similar reduction in inflammatory responses happens in non-vaccinated pregnant women. In contrast, in the vaccinated pregnant women, inflammatory responses are intact. This helps the vaccinated women overcome the virus far better than non-vaccinated participants. Inflammatory responses in vaccinated pregnant women are probably a result of an increased number of effector, memory and naïve CD4 and CD8 T cells. SARS-CoV-2 modifies the T cells and reduces different subtypes of T cells [42,43]. An increase in mesangial cell proliferation that is involved in producing pro-inflammatory cytokines, such as TNF, IL-1 and IL-6, is due to the study period, between 2022 January and February. Omicron BA.1 or BA.2 was circulating, causing very mild illness or asymptomatic infections, even in non-vaccinated subjects. Specifically, the breakthrough infection caused mild or asymptomatic infection in vaccinated women, but it caused a booster immunological response with trained innate immunity induced by vaccination.
Regarding the adaptive immune response, we observed higher serum anti-S IgG levels, as well as S-specific T-cell frequency and proliferative response in vaccinated than non-vaccinated mothers, as also reported in other studies [37]. In addition, we observed the predominance of a CD4 + T h 1 profile in vaccinated women, in agreement with the literature. In particular, while all vaccinated women showed the presence of specific IgG antibodies, and almost all specific T cells, the majority of non-vaccinated women did not show detectable anti-S antibodies or T cells. Women were tested very close to the infection diagnosis after a median time of 2 (0-20) days; we observed, in a previous study of immune response to SARS-CoV-2 infection in the pre-vaccination era, that most infected pregnant women did not show anti-S or anti-NCP IgG within this time frame [44]. Therefore, the presence of both IgG antibodies and T cells against S in the women experiencing SARS-CoV-2 infection after vaccination could be due to the persistence of a vaccine-elicited immune response or to a more rapid response occurring in vaccinated women or both. The cellular immunity following infection or vaccination appears to remain robust and sustained [45], unlike the antibody response that tends to decrease. However, notwithstanding the decrease in antibody levels, memory B cells persists at a stable level for months after vaccination [46], therefore, being able to generate rapidly new antibody-producing cells after a re-challenge by SARS-CoV-2 infection. Of note, the single non-vaccinated women with a previous SARS-CoV-2 infection did not show anti-S IgG antibodies or T cells early after the new infectious episode, conversely to what is instead observed in the vaccinated group. This may suggest that, in some cases, the immunological memory induced by natural infection vaccination may be less sustained than that induced by the mRNA vaccine [47].
All the enrolled women had an asymptomatic or mildly symptomatic infection; therefore, our study cannot provide information about the potentially different clinical manifestation of SARS-CoV-2 infection occurring in vaccinated rather than non-vaccinated pregnant women. However, a clear advantage observed in vaccinated pregnant women developing SARS-CoV-2 infection in the last part of pregnancy is the trans-placental transfer of maternal IgG to the newborn, which was observed in all NBs from vaccinated women and in less than one-third of the newborns from non-vaccinated women. These antibodies may contribute to the protection of NBs in the case of SARS-CoV-2 infection in the first months of life.
As reported in many studies, SARS-CoV-2-specific IgG antibodies are transferred across the placenta to the neonates from their mothers, following SARS-CoV-2 infection [48][49][50]. In particular, women infected in the second trimester (13-26 weeks of gestation) developed antibodies that remained elevated at delivery [19].
Robust IgG levels were noted in all vaccinated pregnant women, and vaccine-induced IgG was transferred to the fetus, as has been noted in the setting of influenza, pertussis and other vaccinations in pregnancy [51,52]. De Rose and colleagues reported a good maternal immune response, as well as the transfer of maternal antibodies to confer passive protection against SARS-CoV-2 in newborns following maternal vaccination [53,54]. Moreover, maternal immunization during the early third trimester (27-31 weeks of gestation) yielded higher neonatal antibody concentrations, compared with the late third trimester (32-36 weeks of gestation) [55,56]. The transfer of maternal antibodies was also noted in breast milk of vaccinated mothers, suggesting a possible specific protective effect on NBs [23,37].
In our study, most non-vaccinated mothers infected in the late third trimester did not transfer IgG antibodies to the NB, likely because they could not develop a sufficient IgG antibody response early after infection, conversely to what observed in the group of infected women that were vaccinated in the preconceptional time or first trimester.
The limitations of this study are the small sample size, due to limited recruitment time, which was not sufficient to compare the clinical manifestation of SARS-CoV-2 infection in vaccinated or non-vaccinated pregnant women, and the lack of follow-up, in order to study and compare the kinetics of the immune response in the two groups. Due to the low sample size, the small p-values obtained in some analyses should be considered with caution.
In conclusion, the early development of antibodies and T-cell responses induced by previous vaccination, but also the rapid production of anti-inflammatory cytokines, could strengthen the immune and inflammatory response after infection. Furthermore, the maternal IgG antibody trans-placental transfer after SARS-CoV-2 infection is favored in previously vaccinated pregnant women and may protect NBs for several months.

Supplementary Materials:
The following supporting information can be downloaded at https://www. mdpi.com/article/10.3390/pathogens12050664/s1. Figure S1. Correlation between the different cytokines in vaccinated (A) and non-vaccinated women (B) was represented. Low and high correlations are indicated with blue and yellow colors respectively. Figure S2. S-specific CD4+ subsets Th1 (A), Th2 (B), and Th17 (C) T cell response. Red dots indicate the women with a previous SARS-CoV-2 infection. Table S1. A factor analysis of the unvaccinated participants. Based on the Eigenvalues, cytokines were classified into four factors. Table S2. Factor analysis of the vaccinated participants. Based on the Eigenvalues, cytokines were classified into five factors. Table S3. Total number of IFN-Y; producing cells for Spike peptide pool, negative control (medium) and PHA. Spike-specific CD4+, CD8+ T-cell and subsets in cell proliferation assay.  Institutional Review Board Statement: The study was approved by the local Ethics Committee, Area Pavia (protocol code: P-20200046007).
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.