Cervicovaginal levels of human beta defensins during bacterial vaginosis.

AIMS: To compare the cervicovaginal levels of human beta defensin (hBD)-1, 2 and 3 of women according to the status of Nugent-defined bacterial vaginosis (BV).
METHODS: A total of 634 women of reproductive age were included in the study. Participants were equally distributed in two groups: according to the classification of vaginal smears according to Nugent criteria in normal (scores 0 to 3) and BV (scores ≥7). Cervicovaginal fluid samples were used for measurements of hBDs1, 2 and 3 levels by enzyme-linked immunosorbent assay (ELISA). Levels of each hBD were compared between the two study groups using Mann-Whitney test, with p-value <0.05 considered as significant. Odds ratio (OR) and 95% confidence interval (95% CI) were calculated for sociodemographic variables and hBD1-3 levels associated with BV a multivariable analysis. Correlation between Nugent score and measured levels of hBDs1-3 were calculated using Spearman's test.
RESULTS: Cervicovaginal fluids from women with BV showed lower levels of hBD-1 [median 2,400.00 pg/mL (0-27,800.00); p<0.0001], hBD-2 [5,600.00 pg/mL (0-45,800.00); p<0.0001] and hBD-3 [1,600.00 pg/mL (0-81,700.00); p = 0.012] when compared to optimal microbiota [hBD-1: [median 3,400.00 pg/mL (0-35,600.00), hBD-2: 12,300.00 pg/mL (0-95,300.00) and hBD-3: 3,000.00 pg/mL (0-64,300.00), respectively]. Multivariable analysis showed that lower levels of hBD-1 (OR: 2.05; 95% CI: 1.46-2.87), hBD-2 (OR: 1.85; 95% CI: 1.32-2.60) and hBD-3 (OR: 1.90; 95% CI: 1.37-2.64) were independently associated BV. Significant negative correlations were observed between Nugent scores and cervicovaginal levels of hBD-1 (Spearman's rho = -0.2118; p = 0.0001) and hBD-2 (*Spearman's rho = -0.2117; p = 0.0001).
CONCLUSIONS: Bacterial vaginosis is associated with lower cervicovaginal levels of hBDs1-3 in reproductive-aged women.

Introduction

Human beta defensins (hBDs) are antimicrobial peptides that have an important role in the innate immune response. They are small cationic peptides produced by epithelial and immunity cells. These peptides act by causing electrostatic imbalance leading to pores formation in the microbial membranes and ultimately cell lysis [1]. Still, hBDs have an indirect lysis mechanism by activating microbial intracellular hydrolases that degrade bacterial cell wall or interfere with membrane lipid distribution [2, 3]. Additionally, hBDs interconnect innate and adaptive immune responses of the host through chemotactic action to T-cells, monocytes, dendritic cells and mast cells, and induce the production of proinflammatory cytokines [2, 4, 5]. There are seventeen types of hBDs identified, of which the most studied are hBD-1, -2 and -3 [4-6]. hBD-1 is recognized as the most important antimicrobial peptide of epithelial cells. Its expression is either constitutive or regulated by inflammation in defense against Gram-negative bacteria [7-9]. hBD-2 is effective against Gram-negative and -positive bacteria [10]. Production of hBD-2 is activated by nuclear Factor kappa B (NF-kappa B), signal transducer and activator of transcription 1 (STAT-1) and activator protein 1 (AP-1) transcription factors [11-13]. hBD-3 is also effective against Gram-negative and -positive bacteria [2, 8], and its production is induced by STAT-1 [12] and AP-1 [14, 15] transcription factors, while hBD-3 activation via NF-kappa B is still controversial [12, 14, 15]. In the context of the female genital tract, hBDs contribute for the protection of the vaginal environment against potential pathogens [16] and vaginal epithelial cells are important sources of hBDs [10, 17, 18].

In normal conditions, Lactobacillus spp. are the main components of vaginal microbiota. Lactobacillus spp. produce lactic acid that leads to an acidic vaginal environment preventing the growth of other bacterial species [19]. In a non-optimal vaginal microbiota Lactobacillus spp. are replaced by other bacterial species, mainly facultative and strict anaerobes. Bacterial vaginosis (BV) is the most frequent type of non-optimal vaginal microbiota and it is often diagnosed by microscopic classification of vaginal smears according to Nugent scoring criteria [20]. Prevalence of BV differs across different populations, affecting 30% to 50% of reproductive-aged women worldwide [21-23].

Up to date, few studies have investigated the relation between cervicovaginal levels of hBDs and the components of vaginal microbiota. In the study by Valore et al. [16] vaginal content of antimicrobial polypeptides was found to be strongly related to the capacity of selectively allowing Lactobacillus growth and inhibiting other bacterial types. However, the few studies available show conflicting results on cervicovaginal levels of hBDs in relation to BV-status. Thus, many aspects of this relation remain unknown. Given the importance of the maintenance healthy vaginal environment for women´s reproductive health [24, 25], the aim of this study was to compare cervicovaginal levels of hBD-1, -2 and -3 between women with Nugent-BV and those with optimal Lactobacillus-dominated microbiota.

Materials and methods

Participants and sample collection

This cross-sectional study was performed with 1096 reproductive-aged women who sought Primary Health Care Units in Botucatu, SP, Brazil, for a routine Pap test from September 2012 to January 2013. Eligibility criteria for inclusion in the study were as follows: non-pregnant, non-menopausal, no hysterectomy, no prior report of HIV seroconversion, no use of hormonal or copper intrauterine device (IUD), no vaginal bleeding, no urinary loss, no use of antibiotics or vaginal cream in the preceding 30 days and abstinence from sexual intercourse in the 72 hours preceding the visit. Written informed consent was obtained from all participants prior to enrollment. The study was approved by the Ethics Committee at the Botucatu Medical School (Protocol 02381512.5.1001.5411).

Data on sociodemographic, sexual behavior and gynecological history of participants were obtained by interview. During physical exam, samples were obtained from the mid-vaginal wall using a sterile cotton swab. These samples were smeared on glass slides for microscopic classification according to Nugent’s scoring system, in normal (scores 0–3), intermediate (scores 4–6) and BV (scores 7–10) [20], and to detect the presence of Candida sp. morphotypes. Samples from the posterior fornix of the vagina were collected to determine infection by Trichomonas vaginalis using culture in Diamond´s medium, and endocervical samples were used for detection of Chlamydia trachomatis and Neisseria gonorrhoeae by PCR. Lastly, 3 mL of sterile saline was used to wash the cervicovaginal region and the liquid was recovered using sterile plastic pipettes. Cervicovaginal fluid samples were centrifuged at 3,000 rpm for 10 min at 4°C and the supernatant was stored at -80°C until hBD-1, -2 and -3 assays.

Detection of Chlamydia trachomatis, Neisseria gonorrhoeae and Trichomonas vaginalis

DNA from cervical samples was extracted using an AmpliLute Liquid Media Extraction Kit (Roche Molecular Systems, Inc.) according to manufacturer´s instructions. C. trachomatis and N. gonorrhoeae were detected by polymerase chain reaction, as previously described [26, 27]. Infection by T. vaginalis was investigated by culture of vaginal posterior fornix samples in Modified Diamond’s medium at 37°C in 5% CO2. Using a clean glass slide without cover and a sterile pipette, fresh wet-mount microscope slides were prepared with aliquots from the culture and examined microscopically under x40 objective. Presence of the motile protozoan was checked daily up to 3 days by the same observer if T. vaginalis motile protozoan was not visualized during this period, the specimen was considered negative.

Quantification of cervicovaginal levels of hBDs

Cervicovaginal fluid samples were evaluated by ELISA using PeproTech specific kits (Rocky Hill, NJ, USA) to measure hBD-1 (cat#900-M202), hBD-2 (cat#900-M172) and hBD-3 (cat#900-M210) levels, following the manufacturer’s instructions. All samples were tested in duplicate. Samples in which hBD levels were estimated to be below the sensitivity of the assay were set as zero, and those with concentrations at levels above standard curve were diluted and re-assayed. The assays were analyzed in an automatic microplate reader (Epoch-BioTek, Winooski, VT, USA), at a wavelength of 492 nm. Intra- and inter-assay coefficients of variation were <5% in this study. Internal laboratory quality-controls assurance program including external and internal standards was followed for all analysis. Minimum detectable levels were 60.00 pg/mL, 130.00 pg/mL, and 4.00 pg/mL for hBD-1, hBD-2 and hBD-3, respectively.

Participant selection and constitution of the study groups

Of the 1096 participants initially included, 37 (3.4%) were excluded by presenting C. trachomatis and 10 (0.9%) for T. vaginalis. None of the participants tested positive for N. gonorrhoeae. Moreover, 112 cases were excluded as microscopic analysis of vaginal microbiota showed other alteration besides BV, as follows: 39 (3.6%) presence of Candida spp. morphotypes, 5 (0.5%) concomitant BV and Candida spp., 62 (5.6%) intermediate microbiota, 6 (0.6%) poor sample quality. Among the 937 remaining participants, 317 were diagnosed with BV and were assigned the study group ´BV´. In order to constitute the control group ´optimal microbiota´, same number of participants (n = 317) were randomly selected amongst the 620 participants that had normal vaginal microbiota.

Results of continuous variables were compared between the two study groups using Mann-Whitney non-parametric test, while those from categorical variables were compared by Chi-square test. P-value inferior to 0.05 considered statistically significant. A multivariable logistic regression analysis was performed using a forward stepwise model selection process (variables retained at P-value ≤0.15) to test the variables independently associated with BV from sociodemographic and hBD1-3 data available. For this analysis we considered as ‘low’ hBDs levels those inferior to the median (2,795.00 pg/mL, 8,335.00 pg/mL and 2,054.00 pg/mL for hBD-1, 2 and 3, respectively). The correlation between Nugent score and measured levels of hBDs1-3 were assessed statistically by calculating Spearman correlation coefficients (Spearman’s rho), for those participants from BV group. Statistical analyses were performed using Stata/SE 15.1 (StataCorp, College Station, TX).

Results

Participant characteristics and clinical history are presented in Table 1. The variables age, marital status, ethnicity, educational status, sexual behavior, vaginal intercourse in the last week and phase of menstrual cycle did not differ between the two study groups. However, the proportion of women that used hormonal contraceptive was significantly higher in optimal microbiota (p = 0.04). Additionally, smoking and higher vaginal pH were more frequent in BV (p = 0.03 and p<0.0001, respectively).

Table 1

Sociodemographic, behavior and clinical characteristics of study population, according to the status of the vaginal microbiota.

Variables	Optimal microbiota (n = 317)	Bacterial vaginosis (n = 317)	p-value
Age a	35 (16–53)	35 (14–54)	0.91
Marital status b
Single	238 (75.1)	220 (69.4)	0.13
In a steady relationship	79 (24.9)	97 (30.6)
Ethnicity (self-reported) b
White	196 (61.8)	193 (60.9)	0.87
Non-white	121 (38.2)	124 (39.1)
Years at school a	11 (0–18)	10 (0–17)	0.28
Number of sex partners, (last 12 months) b
0 or 1	301 (94.5)	294 (92.8)	0.32
2 or more	16 (5.5)	23 (7.2)
Consistent condom use b
Yes	48 (15.2)	49 (15.4)	1.00
No	269 (84.8)	268 (84.6)
Number of vaginal intercourse/week b
0	28 (8.9)	29 (9.1)	0.81
1–2	152 (47.9)	159 (50.2)
3+	137 (43.2)	129 (40.7)
Hormonal contraceptive current use b
Yes	131 (41.3)	105 (33.1)	0.04
No	186 (58.7)	212 (66.9)
Menstrual cycle phases b
Follicular	84(26.5)	97 (30.6)	0.47
Ovulation	16 (5.0)	13 (4.1)
Luteal	217 (68.5)	207 (65.3)
Smoking habit b
Yes	43 (13.6)	64 (20.2)	0.03
No	274 (86.4)	253 (79.8)
Vaginal pH a	4.4 (4.0–5.0)	4.7 (4.0–7.0)	<0.0001

a median (minimum—maximum), non-parametric Mann-Whitney U test.

b n (%), Chi-square test.

The results of cervicovaginal hBDs quantification showed that approximately 10% of the samples presented levels below the limit of detection for hBD-1 and 2. Levels of hBD-3 were undetected in nearly 20% of the sample from both study groups. Fig 1 shows the comparisons of hBD levels between BV and optimal microbiota. Significantly lower cervicovaginal levels of all the three hBDs were observed in BV in relation to optimal microbiota, respectively: hBD-1: 2,400.00 pg/mL (0–27,800.00) vs. 3,400.00 pg/mL (0–35,600.00), p<0.0001; hBD-2: 5,600.00 pg/mL (0–45,800.00) vs. 12,300.00 pg/mL (0–95,300.00), p<0.0001; hBD-3: 1,600.00 pg/mL (0–81,700.00) vs. 3,000.00 pg/mL (0–64,300.00), p = 0.012.

Fig 1

Levels of human beta defensin (hBD)-1, 2 and 3 in cervicovaginal fluid samples of women with bacterial vaginosis (n = 317) compared to those with optimal microbiota (n = 317).

Horizontal bars represent median values. Mann-Whitney U test, p < 0.05.

Multivariable analysis showed that sex partners in the previous year (OR: 1.47 95% CI: 1.06–2.05), and lower levels of hBD-1 (OR: 2.05; 95% CI: 1.46–2.87), hBD-2 (OR: 1.85; 95% CI: 1.32–2.60) and hBD-3 (OR: 1.90; 95% CI: 1.37–2.64) were overrepresented among BV-positive women. Women who reported using hormonal contraceptive were less likely to have BV (OR: 0.55; 95% CI 0.39–0.78) (Table 2).

Table 2

Odds ratio (OR) and 95% confidence intervals (95% CI) for the association between sociodemographic, and low human beta-defensis (hBDs) 1 to 3 cervicovaginal levels and Nugent-bacterial vaginosis.

		Multivariable analysis
		OR	95% CI	P-value
Age		--	--	--
	<35 years
	≥35 years
Marital status		--	--	--
	Single
	In a steady relationship
Ethnicity		--	--	--
	White
	Non-white
Education level		--	--	--
	Uncompleted high school
	Completed high school or above
Number of sex partner (last 12 months)				0.022
	0 to 2	1.47	1.06–2.05
	3 or more
Consistent condom use		--	--	--
	Yes
	No
Frequency of sexual intercourse (per week)		--	--	--
	0 to 2
	3 or more
Hormonal contraceptive use (current)				0.001
	Yes	0.55	0.39–0.78
	No	1.00	--
Phase of menstrual cycle		--	--	--
	Follicular
	Luteal/ovulatory/continous hormonal contraceptive
Smoking habit		--	--	--
	Yes
	No
Low hBD-1 level *				<0.0001
	Yes	2.05	1.46–2.87
	No	1.00	--
Low hBD-2 level *				<0.0001
	Yes	1.85	1.32–2.60
	No	1.00	--
Low hBD-3 level *				<0.0001
	Yes	1.90	1.37–2.64
	No	1.00	--

*Low levels of HBD were set for values inferior to the median.

There were significant negative correlations between Nugent scores and cervicovaginal levels of hBD-1 (Spearman’s rho = -0.2118; p = 0.0001) and hBD-2 (*Spearman’s rho = -0.2117; p = 0.0001), but not for hBD-3 (Spearman’s rho = -0.0600; p = 0.3181).

Discussion

The association between hBDs and BV in reproductive-aged women has been poorly studied to date, and most of them are based on a very limited sample size [18, 28]. Our study included a total of 634 measured samples and showed that BV has lower levels of hBD-1, -2 and -3.

Regarding sociodemographic, behavior and clinical characteristics, it is documented the importance of several factors as smoking, hormonal status, menstrual cycle period and sexual behavior with increased risk for BV [29-33]. In the current study we observed a significant difference regarding smoking habit and hormonal contraceptive use between BV-positive and -negative women, but when we performed a multivariable analysis only the hormonal contraceptive use remained significant. The latter analysis also showed other factors significantly associated with BV as having 3 or more sex partners in the previous year and lower hBD-1, 2 and 3 levels. Interestingly, the menstrual cycle phases were not significant different between the groups. Corroborating our findings, Valore et al. [17] demonstrated significant lower levels of hBD-1 and -2 in women presenting BV compared to healthy women. Recently, Fichorova et al. [34] reported decrease of hBD-2 in Nugent scores of 4 to 6 and 7 to 10, although cervical immunity detectable within 3 months prior to cervicovaginal infection or dysbiosis showed higher hBD-2 levels. In contrast, Fan et al. [28] reported an increase of vaginal hBD-2 in the presence of BV in non-pregnant women. During pregnancy, Mitchell et al. [35] detected lower vaginal levels of hBD-3 in BV, but no difference was observed regarding hBD-2 levels. On the other hand, a lower level of hBD-2 was reported by Kotani et al. [36] in the first trimester of pregnancy in BV-positive women. Experimental data inducing cervical hBD-3 gene expression in a mouse model of ascending infection-related preterm birth demonstrated that hBD-3 reduces microbial ascension into the pregnant uterine cavity, reducing the frequency of premature deliveries [37]. These findings reinforce that hBDs may be potential candidates for augmenting cervical innate immunity, to prevent ascending infection and to reduce susceptibility to sexually transmitted infections, which is supported by the observations that antimicrobial activity is increased in samples with higher hBD-2 concentrations [38]. Therefore, BV and other vaginal dysbiosis seem to be related to lower hBD production, which is reinforced by the current observations.

According to the literature, it is well established that the presence of Lactobacillus spp. in healthy vaginal microbiota has an important role in competitive exclusion of pathogenic bacteria, competition for nutrients, production of antimicrobial substances, and n of the immune system [24, 39]. In this context, Jiang et al. [40] showed a significant correlation between hBD-2 and hBD-3 and DNA levels of L. jensenii, and hBD-2 and DNA levels of L. crispatus in cervicovaginal lavage samples from healthy women. Accordingly, Kotani et al. [18] reported the Nugent score for Lactobacillus morphotype point was well correlated with hBD-2. Additionally, studies using intestinal epithelial cell cultures demonstrate that Lactobacillus spp. are able to up-regulate hBD-2 production [41, 42] by induction of proinflammatory pathways, such as NF-kappa B and AP-1, as well as MAPKs [41]. Therefore, since our results showed decreased levels of hBD-1, -2 and -3 in the presence of BV, a scenario characterized by the absence of Lactobacillus species, we hypothesized that the optimal microbiota had an important role in human beta defensins production. In this sense, a recent study showed that Lactobacillus surface layer proteins (SLP) stimulate the expression of antimicrobial peptides, specifically, SLP of Lactobacillus helveticus SBT2171 promotes hBD-2 expression by activating c-Jun N-terminal kinase (JNK) signaling via Toll-like receptor (TLR)2 in Caco-2 human colonic epithelial cells [43]. Thus, considering the high diversity of bacterial components of the vaginal microbiota, especially during BV, we acknowledge that our study is limited as Nugent scoring does not allow us to determine which bacterial species are present and what their individual association with hBD1-3.

In addition, once BV is characterized by a complex and heterogeneous bacterial community, it may be that bacterial proteases could degrade hBDs [44], which provides an environment that favors the degradation of natural antimicrobial peptides by the diverse proteases produced by different bacterial species [44]. This environment could explain why, even in the presence of proinflammatory cytokines, such as IL-1β [32], which are able to stimulate hBDs production [45], levels of these antimicrobial peptides in women with BV are lower than in optimal microbiota.

Thus, we may hypothesize that the lower levels of hBD-1, -2 and -3 in the presence of BV could be due to the production of proteases by the highly diverse bacterial community of BV and because of the lack of Lactobacillus species in the vaginal microbiota which could alter the secretion of defensins. However, the mechanisms by which vaginal Lactobacillus spp. and epithelial cells interact to enhance the immune response by increasing hBDs production should be further investigated. Moreover, a multivariate analysis has been carried out to explore the nature of the associations observed in the univariate analysis and the levels of hBD 1–3 remain significantly associated with BV as do the number of sexual partners and use of hormonal contraception. Then, we suggest additional future analyses considering this factors, which might provide mechanistic insights into the associations that we described.

Conclusions

Since women with BV had lower cervicovaginal levels of hBD-1, -2 and -3, compared to women with optimal microbiota, we suggest that the presence of Lactobacillus spp. in the vaginal microbiota plays an important role in the production of hBDs in women of reproductive age.

26 Mar 2021

PONE-D-21-03534

Cervicovaginal levels of human beta defensins during bacterial vaginosis

PLOS ONE

Dear Dr. Guimaraes da Silva,

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The method section should provide more details on the optimization and verification of the HBD assays, in particular for what concerns the lower detection levels and reproducibility.

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Reviewer #1: This study builds on our understanding of changes in immune mediator concentrations associated with bacterial vaginosis (BV), which is a key risk factor for sexually transmitted infection (STI) acquisition, including HIV, and adverse reproductive outcomes. A strength of this work is that a large sample size of 634 women was included. hBDs-1, -2 and -3 were measured in duplicate, another strength, in all samples. Although women with common STIs, intermediate microbiota or candidiasis were excluded, the analysis presented would benefit from multivariate approaches to identify and address other possible confounders and effect modifiers. A clear description of the quality control analysis would also be useful.

1. Although the manuscript is generally clear, concise and flows well, there are (mostly minor) grammatical errors throughout and I would recommend copy editing.

2. Page 6: It is reported that women with C. trachomatis, T. vaginalis, candidiasis or intermediate microbiota were excluded. Although N. gonorrhoeae was assessed, it is not reported whether any women had this infection or if women with this infection were also excluded.

3. Page 6, line 134: “Minimum detectable levels were 0.06 pg/mL, 0.13 pg/mL, and 0.004 pg/mL for hBD-1, hBD-2 and hBD-3, respectively”. This seems very low, particularly for hBD-3. Standard curves shown on the manufacturer’s website show that hBD-3 seems to plateau before 10pg/ml. Can the authors confirm the accuracy of measurements in the <10 pg/ml range?

4. Page 6, line 138: Please clarify the following: “37 (3.4%) were excluded by presenting

C. trachomatis or T. vaginalis (n=10, 0.9%)”. Does the “37” refer to C. trachomatis and the “10” refer to T. vaginalis? This is not written clearly.

5. Were any women using the copper IUD – only hormonal methods and condoms are mentioned?

6. The authors should adjust p-values for multiple comparisons where applicable.

7. Since the hBDs were measured in duplicate, it would be useful to present some data showing the reproducibility of the measurements – e.g. CVs

8. Were hBDs associated with any demographic/socio-behavioural factors assessed in this study? The authors should assess this and evaluate the relationship between hBDs and BV, adjusting for possible confounders and effect modifiers e.g. hormonal contraceptive use and smoking differ between comparison groups.

9. Page 9-11: The following conclusions should be reworded:

“Thus, the production of hBDs is related to the presence of Lactobacillus spp. in the vaginal microbiota.”

“but is mainly due to the lack of Lactobacillus species in the vaginal microbiota which could modulate the secretion of defensins”

“we suggest that the presence of Lactobacillus spp. in the vaginal microbiota plays an important role in the regulation of human beta defensins production in women of reproductive age.”

It is not possible, based on the data presented in this study (i.e. Nugent score) to conclude that hBD production is related to Lactobacillus spp. specifically. The relationship may have nothing to do with the lactobacilli, but may rather be due to downregulation or degradation of hBD production in the presence of non-optimal bacteria (which is discussed on page 11). It would also be useful to compare the score for the Lactobacillus morphotype to hBD concentrations – a sub-analysis could also be conducted to evaluate the relationship between Lactobacillus score and hBD concentrations among BV negative women only (i.e. when Gardnerella and Mobiluncus morphotypes are absent or rare, is hBD associated with Lactobacillus morphotype score?).

10. Page 10, line 217: Please clarify the following statement regarding “activation of the immune system” by Lactobacillus spp. since lactobacilli are also considered immunoregulatory.

11. It would be preferable to refer to “optimal” microbiota, instead of “normal” microbiota. See McKinnon et al. AIDS Research and Human Retroviruses. 2019 Mar 1;35(3):219-28.

Reviewer #2: The authors measured the levels of HBD1, 2 and 3 in CVL from 634 women by BV status defined by Nugent score. They show that the levels of all three defensins to be significantly lower in 317 women with BV (Nugent score of >7) than in 317 randomly selected women without BV (Nugent score 0-3). BV is associated with poor health outcomes and increased susceptibility to HIV which has been associated with pro-inflammatory responses and there is great interest in gaining a better understanding of the etiology.

It is argued that via their direct antimicrobial action, the beta defensins contribute to the protection of the vaginal microenvironment. There is circumstantial evidence that the innate immune response to resident Lactobacilli which includes anti-microbial peptides produced by the vaginal epithelium, contributes to protection against bacterial colonisation and subsequent dysbiosis associated with BV. The authors cite evidence that vaginal antimicrobial polypeptides can selectively allow for the growth of (resident) Lactobacilli, which is suggestive of a mechanism but the data is derived from a small number of women.

Existing data on the association with BV and levels of HBDs is limited and somewhat contradictory with some studies showing higher levels of HBD and some showing lower levels. HBDs recruit innate and adaptive immune cells and associated proinflammatory cytokines such as IL6 and IL8 have also been measured in some studies- again with conflicting results. In a review from 2014 which is not cited by the authors, Mitchell and Marazzo discuss the immune response associated with BV and any evidence for the modulating impact of Lactobacilli in the context of inflammatory markers (associated with increased susceptibility HIV). They review data on the associations of the proinflammatory cytokines ILbeta and IL8 with BV and also discuss associations with antimicrobial peptides. There is no consistent finding and they conclude that the considerable variability in findings is probably due to the fact that most studies are small and underpowered and constrained by their cross-sectional design which does not account for the acknowledged (almost daily) fluctuations in vaginal microbiota (Gajer 2012). Longitudinal treatment studies are considered more robust and provide more consistent data with 5/6 showing reductions in levels of IL1beta and 3/6 reductions in IL8 after treatment for BV. Data on HBD was derived predominantly from pregnant women (Mitchell et al 2013) and showed that all 9 bacterial species associated with BV were associated with lower levels of HBD3 – but this was not true for HBD2. In a more direct approach, genetic analysis has also shed some light on factors associated with susceptibility to BV with polymorphisms in TLR IL6 and IL8 associated with increased susceptibility to BV arguing a role a robust innate immune response in protection. Whilst data on cytokines and BV has also been conflicting, this reviewer thinks that the paper would be strengthened by inclusion of data on the levels of proinflammatory cytokines such as IL1b, IL6 and IL8 as this would provide insights into the immunological consequences of the lower observed levels of HBD1, 2, 3 and potentially provide some mechanistic insights which have been suggested.

The etiology of BV is undoubtedly complex and multifactorial –without even considering the diversity in bacterial species which have been associated with both health and disease across different studies which complicates the interpretation of associations seen. The authors have not addressed this complexity and the gaps in their review of the literature reflects this. In their review Martin and Marazzo (JID 2014) discuss the importance of accounting for smoking, hormonal status and sexual behaviour in analyses as these are all known to play a role (Mitchell and Murphy 2014). In particular, estrodiol has been shown to be reduce the production of HBD2 and SLP and mRNA expression of TLR by vaginal epithelial cells in vitro (Wira et al 2015). In their comprehensive paper of 2019, Fichorova et al also describe an important role for hormonal contraception in susceptibility to dysbiosis in the context of innate immune responses. Inflammatory responses are also known to fluctuate throughout the menstrual cycle with lower levels from day 7-10. These authors show that levels of smoking and hormonal contraceptive use differ significantly by BV status and this could confound their analysis. The authors state that they cannot access any more information about hormonal contraceptive use, but they should at least discuss the limitations of their study in the context of available literature. At the very least they should run a multivariate analysis to accounts for smoking and hormonal status to see whether the associations with BV and HBD remain significant.

It is acknowledged that BV is characterised by the general absence of “healthy Lactobacilli “ and it is debated whether the lactobacilli exert a positively protective effect. It has been shown for example that L crispatus and L jensenii are able to directly induce the production of HBD2 and thereby inhibit the growth of some gram negative bacteria. The reviewer understands that it is probably beyond the scope of the authors to undertake a comprehensive characterisation of the bacterial composition within their cohort, but this should be addressed within the discussion.

In conclusion, this cross sectional study provides some interesting suggestive evidence that anti-microbial defensins might be associated with BV but limitations in the design mean that it provides limited additional extra value to the existing literature in its current form . The paper would be benefit from additional analysis to account for known confounders,a more thorough review of the literature discussing limitations, and if at all possible inclusion of additional data on levels of the pro-inflammatory cytokines which have been shown to be associated with BV.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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28 Jul 2021

RESPONSE TO REVIEWERS

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

________________________________________

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: No

________________________________________

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This study builds on our understanding of changes in immune mediator concentrations associated with bacterial vaginosis (BV), which is a key risk factor for sexually transmitted infection (STI) acquisition, including HIV, and adverse reproductive outcomes. A strength of this work is that a large sample size of 634 women was included. hBDs-1, -2 and -3 were measured in duplicate, another strength, in all samples. Although women with common STIs, intermediate microbiota or candidiasis were excluded, the analysis presented would benefit from multivariate approaches to identify and address other possible confounders and effect modifiers. A clear description of the quality control analysis would also be useful.

Answer: We appreciate the careful review and enriching comments made by this reviewer. All points were discussed by the authors and answers and comments are provided bellow.

1. Although the manuscript is generally clear, concise and flows well, there are (mostly minor) grammatical errors throughout and I would recommend copy editing.

Answer: In fact we have detected some writing errors and have rewritten according.

2. Page 6: It is reported that women with C. trachomatis, T. vaginalis, candidiasis or intermediate microbiota were excluded. Although N. gonorrhoeae was assessed, it is not reported whether any women had this infection or if women with this infection were also excluded.

Answer: We thank this reviewer for pointing that out. None of the participants tested positive for Neisseria gonorrhoeae, as now stated in page 6 (results section).

3. Page 6, line 134: “Minimum detectable levels were 0.06 pg/mL, 0.13 pg/mL, and 0.004 pg/mL for hBD-1, hBD-2 and hBD-3, respectively”. This seems very low, particularly for hBD-3. Standard curves shown on the manufacturer’s website show that hBD-3 seems to plateau before 10pg/ml. Can the authors confirm the accuracy of measurements in the <10 pg/ml range?

Answer: We thank the reviewer for this pointing this out. We did not realize that the measure unity was in ng/mL instead of pg/mL. Therefore, we corrected the data multiplying the measured values by a factor of 1000 so it is now correctly expressed in pg/mL. Minimum values are very close to manufacture’s standard curve interval. We regret the submitted manuscript had this mistake and thank the reviewer for the careful analysis. All values were corrected for the newly version of the submitted paper.

4. Page 6, line 138: Please clarify the following: “37 (3.4%) were excluded by presenting

C. trachomatis or T. vaginalis (n=10, 0.9%)”. Does the “37” refer to C. trachomatis and the “10” refer to T. vaginalis? This is not written clearly.

Answer:We thank this reviewer for pointing this issue. In fact, the text was not sufficiently accurate. We corrected the text for “37 (3.4%) were excluded by testing positive for presenting C. trachomatis and 10 (0.9%) for T. vaginalis” (page 06) at the results section of the manuscript.

5. Were any women using the copper IUD – only hormonal methods and condoms are mentioned?

Answer We appreciate this reviewer mentioning the possibility of having IUD-users in our study population. We did not considered for study enrollment those women who reported IUDs of any type (cooper or hormonal), because their known association with BV. We understand that this should be clearly stated in the text. Please see the text added to page 4 (line 106) for addressing this topic.

6. The authors should adjust p-values for multiple comparisons where applicable.

Answer: We do agree with this reviewer that multiple comparisons is relevant and should be included in the analysis. Thus, we performed a multivariable logistic regression analysis using a forward stepwise model selection process (variables retained at P-value ≤0.15) to identify sociodemographic and hBDs variables independently associated with BV from sociodemographic and hBD1-3 data available. This analysis is presented in Results section pages 09-10.

7. Since the hBDs were measured in duplicate, it would be useful to present some data showing the reproducibility of the measurements – e.g. CVs

Answer: We do agree with this reviewer that this information is relevant and should be included in the text. We have added this information at the methods section in page 06. We correct the text including this information “Intra- and inter-assay coefficients of variation were <5% in this study.

8. Were hBDs associated with any demographic/socio-behavioural factors assessed in this study? The authors should assess this and evaluate the relationship between hBDs and BV, adjusting for possible confounders and effect modifiers e.g. hormonal contraceptive use and smoking differ between comparison groups.

Answer: We do agree with this reviewer that evaluate the relationship between hBDs and BV, adjusting for possible confounders. Please refer to the answer provided to question number 6.

9. Page 9-11: The following conclusions should be reworded:

“Thus, the production of hBDs is related to the presence of Lactobacillus spp. in the vaginal microbiota.”

“but is mainly due to the lack of Lactobacillus species in the vaginal microbiota which could modulate the secretion of defensins”

“we suggest that the presence of Lactobacillus spp. in the vaginal microbiota plays an important role in the regulation of human beta defensins production in women of reproductive age.”

It is not possible, based on the data presented in this study (i.e. Nugent score) to conclude that hBD production is related to Lactobacillus spp. specifically. The relationship may have nothing to do with the lactobacilli, but may rather be due to downregulation or degradation of hBD production in the presence of non-optimal bacteria (which is discussed on page 11). It would also be useful to compare the score for the Lactobacillus morphotype to hBD concentrations – a sub-analysis could also be conducted to evaluate the relationship between Lactobacillus score and hBD concentrations among BV negative women only (i.e. when Gardnerella and Mobiluncus morphotypes are absent or rare, is hBD associated with Lactobacillus morphotype score?).

Answer: We do agree with this reviewer that it is not possible, based on the data presented in this study (i.e. Nugent score) to conclude that hBD production is related to Lactobacillus spp. specifically. Then, as suggested, we performed the correlation between Nugent score and measured levels of hBDs1-3 were assessed statistically by calculating Spearman correlation coefficients (Spearman’s rho), for those participants from BV group. This analysis is presented in Results section page 10.

10. Page 10, line 217: Please clarify the following statement regarding “activation of the immune system” by Lactobacillus spp. since lactobacilli are also considered immunoregulatory.

Answer:We thank this reviewer for pointing this issue. In fact, the text was not clear. We corrected the text for “and tolerance of the immune system” (page 12).

11. It would be preferable to refer to “optimal” microbiota, instead of “normal” microbiota. See McKinnon et al. AIDS Research and Human Retroviruses. 2019 Mar 1;35(3):219-28.

Answer: We replaced “normal” microbiota for “optimal microbiota” throughout the manuscript.

Reviewer #2: The authors measured the levels of HBD1, 2 and 3 in CVL from 634 women by BV status defined by Nugent score. They show that the levels of all three defensins to be significantly lower in 317 women with BV (Nugent score of >7) than in 317 randomly selected women without BV (Nugent score 0-3). BV is associated with poor health outcomes and increased susceptibility to HIV which has been associated with pro-inflammatory responses and there is great interest in gaining a better understanding of the etiology.

It is argued that via their direct antimicrobial action, the beta defensins contribute to the protection of the vaginal microenvironment. There is circumstantial evidence that the innate immune response to resident Lactobacilli which includes anti-microbial peptides produced by the vaginal epithelium, contributes to protection against bacterial colonisation and subsequent dysbiosis associated with BV. The authors cite evidence that vaginal antimicrobial polypeptides can selectively allow for the growth of (resident) Lactobacilli, which is suggestive of a mechanism but the data is derived from a small number of women.

Existing data on the association with BV and levels of HBDs is limited and somewhat contradictory with some studies showing higher levels of HBD and some showing lower levels. HBDs recruit innate and adaptive immune cells and associated proinflammatory cytokines such as IL6 and IL8 have also been measured in some studies- again with conflicting results. In a review from 2014 which is not cited by the authors, Mitchell and Marazzo discuss the immune response associated with BV and any evidence for the modulating impact of Lactobacilli in the context of inflammatory markers (associated with increased susceptibility HIV).

Answer:We agree with this reviewer that this review is important in this context and we included it in the discussion, page 11.

They review data on the associations of the proinflammatory cytokines ILbeta and IL8 with BV and also discuss associations with antimicrobial peptides. There is no consistent finding and they conclude that the considerable variability in findings is probably due to the fact that most studies are small and underpowered and constrained by their cross-sectional design which does not account for the acknowledged (almost daily) fluctuations in vaginal microbiota (Gajer 2012). Longitudinal treatment studies are considered more robust and provide more consistent data with 5/6 showing reductions in levels of IL1beta and 3/6 reductions in IL8 after treatment for BV. Data on HBD was derived predominantly from pregnant women (Mitchell et al 2013) and showed that all 9 bacterial species associated with BV were associated with lower levels of HBD3 – but this was not true for HBD2. In a more direct approach, genetic analysis has also shed some light on factors associated with susceptibility to BV with polymorphisms in TLR IL6 and IL8 associated with increased susceptibility to BV arguing a role a robust innate immune response in protection. Whilst data on cytokines and BV has also been conflicting, this reviewer thinks that the paper would be strengthened by inclusion of data on the levels of proinflammatory cytokines such as IL1b, IL6 and IL8 (temos n=163 com IL1 e IL6 apenas para microbiota normal) as this would provide insights into the immunological consequences of the lower observed levels of HBD1, 2, 3 and potentially provide some mechanistic insights which have been suggested.

Answer: We appreciate this point out by the reviewer and we agree that it will be an excellent opportunity for a new study, but, at this time, unfortunately we can`t assess this cytokines because most of the cervicovaginal samples were depleted during hBDs assays.

The etiology of BV is undoubtedly complex and multifactorial –without even considering the diversity in bacterial species which have been associated with both health and disease across different studies which complicates the interpretation of associations seen. The authors have not addressed this complexity and the gaps in their review of the literature reflects this (incluir diversidade da vb e que nossa forma de acessar a microbiota noa permite detectar essa heterogeneidade da VB). In their review Martin and Marazzo (JID 2014) discuss the importance of accounting for smoking, hormonal status and sexual behaviour in analyses (se basear nisso para fazer as subanalises da comparacao dos ,niveis de hbds entre os grupos) as these are all known to play a role (Mitchell and Murphy 2014). In particular, estrodiol has been shown to be reduce the production of HBD2 and SLP and mRNA expression of TLR by vaginal epithelial cells in vitro (Wira et al 2015) (citar isso na discussao). In their comprehensive paper of 2019, Fichorova et al also describe an important role for hormonal contraception in susceptibility to dysbiosis in the context of innate immune responses. Inflammatory responses are also known to fluctuate throughout the menstrual cycle with lower levels from day 7-10.

Answer: All studies suggested by this reviewer were included in the discussion of the current version of the manuscript. We do appreciate all these enriching suggestions. In addition, we regret to inform that we cannot assess cytokines levels because samples were depleted. We do agree with these reviewer and consider this is an excellent opportunity for a new study.

These authors show that levels of smoking and hormonal contraceptive use differ significantly by BV status and this could confound their analysis. The authors state that they cannot access any more information about hormonal contraceptive use, but they should at least discuss the limitations of their study in the context of available literature. At the very least they should run a multivariate analysis to accounts for smoking and hormonal status to see whether the associations with BV and HBD remain significant.

Answer: We do agree with this reviewer that evaluate the association between hBDs and BV, adjusting for possible confounders is relevant and should be included in the results. Thus, we performed a multivariable logistic regression analysis using a forward stepwise model selection process (variables retained at P-value ≤0.15) to test the variables independently associated with BV from sociodemographic and hBD1-3 data available. This analysis is presented in Results section pages 09-10.

It is acknowledged that BV is characterised by the general absence of “healthy Lactobacilli “ and it is debated whether the lactobacilli exert a positively protective effect. It has been shown for example that L crispatus and L jensenii are able to directly induce the production of HBD2 and thereby inhibit the growth of some gram negative bacteria. The reviewer understands that it is probably beyond the scope of the authors to undertake a comprehensive characterisation of the bacterial composition within their cohort, but this should be addressed within the discussion.

Answer: We thank this reviewer for this interesting comment. We do agree that Nugent scoring does not allow the full characterization of the microbiota and should be acknowledged as a study limitation. Text added to the discussion section at pages 12-13)

In conclusion, this cross sectional study provides some interesting suggestive evidence that anti-microbial defensins might be associated with BV but limitations in the design mean that it provides limited additional extra value to the existing literature in its current form . The paper would be benefit from additional analysis to account for known confounders,a more thorough review of the literature discussing limitations, and if at all possible inclusion of additional data on levels of the pro-inflammatory cytokines which have been shown to be associated with BV.

Answer: We thank this reviewer for acknowledging our efforts. We do agree that the current studies has limitations (i. e. no cytokine data), but it should contribute with robust data that may contribute for better understanding the relation between BV and cervicovaginal hBDs.

________________________________________

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Submitted filename: Response to reviewers.docx

Click here for additional data file.

28 Oct 2021

PONE-D-21-03534R1Cervicovaginal levels of human beta defensins during bacterial vaginosisPLOS ONE

Dear Dr. Guimaraes da Silva,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

I feel that the manuscript's quality has been improved, but there are still some  inconsistencies to be fixed, as suggested by one of the referees Please submit your revised manuscript by Dec 12 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Kind regards,

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Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Partly

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: I Don't Know

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: The manuscript is considerably improved and the authors have addressed many of the editorial comments. There are still some careless inconsistencies throughout and these should be addressed with thought for the overall flow of the discussion.

A major strength of the study lies in the number of individuals which have been included in the analysis, but the interpretation of the associations seen is hampered by the limited analysis performed so the authors should be careful not to over interpret their findings.

Specifically, the authors should caution against drawing firm conclusions about the association Lactobacilli spp and levels of hBD as they have not characterised the bacterial composition of the swabs. Statements such as

line 224 “ Thus, the production presence of hBDs is related to the presence of a increased abundance of Lactobacillus spp. in the vaginal microbiota “

should be modified accordingly to be more consistent with

line 283 “Thus, we postulate may hypothesize that the decreased lower levels of hBD-1, -2 and -3 in the presence of BV could be, in part, due to the production of proteases by the complex highly diverse bacterial community of BV,”

Importantly, a multivariate analysis has been carried out to explore the nature of the associations observed in the univariate analysis. The levels of HBD 1-3 remain significantly associated with BV as do the number of sexual partners and use of hormonal contraception. The authors discuss their findings in the context of previous data, and perhaps could suggest additional future analyses in their discussion which might provide mechanistic insights into the associations that they describe (line 289)

There remain some careless “typos”;

Line 172 The concentrations of hBD are still 1000X too low. These errors have been corrected elsewhere in the manuscript and this is a careless oversight.

Reviewer #3: The manuscript "Cervicovaginal levels of human beta defensins during bacterial vaginosis" addresses a complex and controversial issue that has been under research thoroughly for the last years: the relationship between vaginal microbiota and endogenous inflammatory factors and regulation.

I agreed with most of the comments and observations of the two previous reviewers and found that the authors have addressed them appropriately. After the corrections and additions made, the manuscript has significantly improved.

Even when the methodology used is simple and does not allow a more profound analysis (such as characterization of Lactobacillus species), the conclusions are valuable and contribute to this field's knowledge.

Thus, it's my recommendation to accept it for publication.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

3 Nov 2021

RESPONSE TO REVIEWERS

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Answer: We found a typo in the title of reference 45 which was corrected, and some formatting errors along the references which were corrected as well. We did not cite papers retracted.

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

________________________________________

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Partly

Reviewer #3: Yes

________________________________________

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: I Don't Know

________________________________________

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: Yes

________________________________________

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: Yes

________________________________________

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: The manuscript is considerably improved and the authors have addressed many of the editorial comments. There are still some careless inconsistencies throughout and these should be addressed with thought for the overall flow of the discussion.

A major strength of the study lies in the number of individuals which have been included in the analysis, but the interpretation of the associations seen is hampered by the limited analysis performed so the authors should be careful not to over interpret their findings.

Specifically, the authors should caution against drawing firm conclusions about the association Lactobacilli spp and levels of hBD as they have not characterised the bacterial composition of the swabs. Statements such as

line 224 “ Thus, the production presence of hBDs is related to the presence of a increased abundance of Lactobacillus spp. in the vaginal microbiota “

should be modified accordingly to be more consistent with

line 283 “Thus, we postulate may hypothesize that the decreased lower levels of hBD-1, -2 and -3 in the presence of BV could be, in part, due to the production of proteases by the complex highly diverse bacterial community of BV,”

Answer: We thank the reviewer for this pointing this out. Then, we removed this sentence “ Thus, the production presence of hBDs is related to the presence of a increased abundance of Lactobacillus spp. in the vaginal microbiota “.

Importantly, a multivariate analysis has been carried out to explore the nature of the associations observed in the univariate analysis. The levels of HBD 1-3 remain significantly associated with BV as do the number of sexual partners and use of hormonal contraception. The authors discuss their findings in the context of previous data, and perhaps could suggest additional future analyses in their discussion which might provide mechanistic insights into the associations that they describe (line 289)

Answer: We do agree with the reviewer that this information is relevant and should be included in the text. We just added this information at the discuss section in last paragraph.

There remain some careless “typos”;

Line 172 The concentrations of hBD are still 1000X too low. These errors have been corrected elsewhere in the manuscript and this is a careless oversight.

Answer: In fact we have detected some typos errors and have rewritten according.

Reviewer #3: The manuscript "Cervicovaginal levels of human beta defensins during bacterial vaginosis" addresses a complex and controversial issue that has been under research thoroughly for the last years: the relationship between vaginal microbiota and endogenous inflammatory factors and regulation.

I agreed with most of the comments and observations of the two previous reviewers and found that the authors have addressed them appropriately. After the corrections and additions made, the manuscript has significantly improved.

Even when the methodology used is simple and does not allow a more profound analysis (such as characterization of Lactobacillus species), the conclusions are valuable and contribute to this field's knowledge.

Thus, it's my recommendation to accept it for publication.

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Reviewer #2: No

Reviewer #3: No

Submitted filename: RESPONSE TO REVIEWERS.pdf

Click here for additional data file.

17 Nov 2021

Cervicovaginal levels of human beta defensins during bacterial vaginosis

PONE-D-21-03534R2

Dear Dr. da Silva,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Additional Editor Comments (optional):

Reviewers' comments:

22 Nov 2021

PONE-D-21-03534R2

Cervicovaginal levels of human beta defensins during bacterial vaginosis

Dear Dr. Silva:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Academic Editor

PLOS ONE