Effectiveness of vaginal probiotics Lactobacillus crispatus chen-01 in women with high-risk HPV infection: a prospective controlled pilot study

Introduction

Human papillomavirus (HPV) infection is one of the most common sexually transmitted diseases of the female lower genital tract worldwide. The prevalence of female genital HPV infection ranges from 12% to 49.5% showing regional variation [1, 2], meanwhile, around 80% of women have the chance to be exposed to HPV during lifetime [3]. Genital HPVs can be subdivided into high- and low-risk types, and persistent high-risk HPV infection gives rise to cervical intraepithelial neoplasia (CIN) or even cervical cancer. It has been reported that almost all invasive cervical cancer biopsies contain intact HPV-DNA [4], thus, eradication of HPV infection is the key to preventing CIN and cervical cancer. Currently, inoculations of HPV vaccine are the main way to prevent HPV infection. However, even the most popular nine-valent vaccine does not fully cover all high-risk types. Therefore, the lack of treatments for those who have been infected with HPV promote the urgency to develop safe and effective anti-HPV methods in the prevention and treatment of cervical cancer.

The vagina is an outside-communicating channel for sexual intercourse and menstruation, which connects upwards to the cervix, and downwards to the external environment. It is colonized by a plethora of microorganisms, including aerobic bacteria, anaerobic bacteria, known as the commensal microbiota, which interact with each other to keep the balance of vaginal microbiota and a healthy state [5]. Lactobacillus is the dominant bacterial species in the reproductive tract of healthy women, which produces lactic acid, hydrogen peroxide, and bacteriocins to maintain an acidic environment and prevent pathogen growth, adheres on the epithelium, repels other bacteria adhesion, and regulates immune and inflammatory response, enhancing the resistance of vagina to diseases [6]. However, vaginal microbiota is susceptible to various influence factors, such as menstrual cycle, sexual activity, and douching [7]. Once the balance is disrupted, it leads to various vaginal inflammatory diseases, such as bacterial vaginosis (BV), vulvovaginal candidiasis (VVC), and sexually transmitted diseases including HPV infection [8, 9].

Recently, increased evidence has suggested that the abnormal vaginal microbiota plays a role in the acquisition and persistence of HPV infection and elevates the risk of cervical cancer [10]. High-throughput sequencing of vaginal secretions revealed significant differences of vaginal microbiota between HPV-negative and HPV-positive women, which manifests as a highly diverse vaginal microbiota with a decrease of Lactobacillus spp. and increase of Gardnerella vaginalis [11]. Normally, Lactobacillus prevents the colonization of species related to bacterial vaginosis by maintaining low pH and producing bacteriocins, which is important for cervical epithelial barrier function of inhibiting HPV from entering basal keratinocytes [12]. When dysbiosis of vaginal microbiota happens, the number of Lactobacilli reduces, and Gardnerella as well as anaerobic bacteria colonize massively and release enzymes and metabolites, which may damage the cervical epithelial barrier and provide opportunities for HPV infection and invasion [11, 13]. HPV infection is closely related to the dysbiosis of local vaginal microecology, especially the reduction of Lactobacilli [14]. Thus, restoring vaginal microecological balance with Lactobacillus supplementation is expected to be a potential and promising therapeutic strategy for HPV infection.

Probiotics are defined as “living microorganisms that can confer health benefits to the host when applied in sufficient quantities” [15], which exert beneficial effects through various mechanisms, including modulating pH, decreasing the colonization and invasion of other pathogenic organisms, as well as modifying the host immune responses [16]. At present, probiotics and their products have been widely used in gastrointestinal disorders, allergic diseases, diabetes, cardiovascular diseases and cancers [17, 18]. In the female reproductive tract, the most common probiotics are Lactobacillus spp., including L. crispatus, L. gasseri, L. iners, and L. jensenii [18]. However, there are few commercial vaginal probiotics currently available in the market. In China, the only available vaginal probiotic is the Living Preparation of Lactobacillus, which is composed of non-human origin L. delbrueckii with low bacterial content. Meanwhile, the application of probiotic products in gynecological diseases is limited, and prescription drugs mainly target the treatment of bacterial vaginosis. Therefore, the potential use of probiotics in gynecological diseases including interventions on HR-HPV infection needs to be further explored.

Considering the scant awareness on the clinical use of probiotics in gynecological diseases, few research on the local transplantation of Lactobacilli for the treatment of gynecological diseases has been reported, potentially hindered by the difficulty in the large sample recruitment and the ethical limitations on the clinical research. Although studies in the animal stage have been proved effective [19], the compositional difference between vaginal microbiota in humans and animals, as well as its correlated mechanism greatly limited the further research. Moreover, since the traditional oral administration route may undergo the first elimination of the liver, and how it adjusts the vaginal microecological balance is still unclear, the mode of administration remains to be considered. Encouragingly, with the development of fecal microbiota transplantation in full swing, local transplantation of Lactobacilli has aroused broad concern. Benefiting from the application of high-throughput sequencing, roles of single-strain probiotics in the treatment of gynecological diseases have also been continuously explored. In this study, we take the lead to testify the role of L. crispatus chen-01, a natural probiotic strain isolated from the healthy human in the intervention of HR-HPV infection. 100 subjects with high-risk HPV infection were recruited, and the clinical safety and efficacy of intravaginal administration of L. crispatus chen-01 were evaluated. High-throughput sequencing was used to analyze the diversity and alteration of vaginal microbial composition. Our study explored the correlation between cervical HR-HPV infection and vaginal microecology, as well as the role of probiotic intervention in the treatment of HR-HPV, which provides a novel strategy for the treatment and management of cervical HR-HPV infection, and a promising concept for the prevention and treatment of cervical intraepithelial neoplasia and cervical cancer.

Results

Discussion

Cervical cancer (CC) is the fourth most common malignancy in women worldwide [20]. It is well established that a persistent infection with high-risk HPV (HR-HPV) causes nearly all cases of invasive cervical cancer [21]. Therefore, eradicating HPV is expected to be an effective way to prevent cervical cancer. Recently, emerging literatures have highlighted the significance of cervicovaginal microbiota in the HR-HPV susceptibility and clearance [22], which indicates regulating cervicovaginal microbiota might be an effective treatment strategy for HP-HPV infection.

Since HR-HPV infection with high-grade cervical intraepithelial neoplasia, cervical cancer or tumors in the genital tract require clinical intervention such as surgical or medical treatment, in order to testify the effectiveness of involving vaginal microbiota in the treatment of HPV infection, we recruited high-risk HPV infection cases mostly without cervical lesions or diagnosed as low-grade cervical intraepithelial neoplasia and administered a natural probiotic stain isolated from the vagina in the healthy individuals—L. crispatus chen-01 locally to observe the alterations of clinical parameters and vaginal microbiota. In the clinical practice, we observed the ameliorative effect of intravaginal transplantation of L. crispatus chen-01 on patients with HR-HPV infection. Our results showed a significant reduction in HPV viral load and a 1.3-fold increase of HPV clearance rate in patients who received L. crispatus transplantation compared with the control group. Although both of the two groups showed reduction of viral load, there are significant difference between the two groups in the follow-up 6th month, which means that the application of probiotics facilitated the clearance of HPV in addition to the natural clearance. Our result is consistent with Pierro’s study that oral administration of L. crispatus could change the vaginal CST status in patients and improve the clearance rate of HPV [23]. Similarly, Dellino et al. also showed that total HPV clearance increase from 9.3% to 15.3% in the patients taking long term oral L. M247 compared with those who didn’t receive following interventions [24]. Vaginal microbiota transplantation could reconstitute healthy vaginal microbiota, which had a certain effect on bacterial vaginosis [25]. Previous research also indicated that the long-term vaginal administration of Lactobacilli in HPV-infected patients with bacterial vaginitis or vaginitis help to eliminate not only vaginitis but also the viral infection [26]. Here, our result further confirmed that the use of vaginal probiotics L. crispatus chen-01 could effectively improve vaginitis and cervical exfoliative cytology without causing severe adverse reactions.

In order to analyze the possible mechanism of HPV clearance by L. crispatus, high-throughput sequencing was used to analyze the diversity of cervicovaginal microbiota and the changes of vaginal microbial composition pre and post treatment of L. crispatus. Recent cross-sectional studies have revealed a higher diversity of cervicovaginal microbiota in HPV-positive women compared with that of HPV-negative individuals [27, 28]. Our analysis further indicated an increase of Prevotella and Gardnerella, but a decrease of Lactobacillus in high-risk HPV cases, which is consistent with the research performed by Dong et al. that the overgrowth of Prevotella may participate in the occurrence of persistent HR-HPV infection-related cervical lesions [29]. Encouragingly, by giving Lactobacillus locally, this trend was expected to be reversed. A healthy vaginal microbiota is mainly composed of 1 - 2 Lactobacillus species, showing low species diversity [5]. Our results showed that probiotic treatment significantly reduced the diversity of vaginal microbiota both at the phylum and genus levels, similar to those of the healthy female.

In the normal female reproductive tract, vaginal microbiota is dominated by Lactobacillus spp., accounting for 70–95% [5]. Lactobacillus-dominated microbiota has long been considered as the hallmark of health in the female reproductive tract [30]. Among this, L. crispatus can inhibit the growth of various vaginal pathogens and fungi, improve antibiotic sensitivity, and reduce the risk of sexually transmitted diseases [31]. These properties of L. crispatus may be related to its capacity to adhere on the host cells to prevent pathogen colonization, produce bactericidal substances (lactic acid, hydrogen peroxide), niche occupancy, and immune regulation [6]. Studies have shown that L. crispatus can promote the proliferation of vaginal epithelial cells and the healing of damaged epithelium, thus maintaining the integrity of the vaginal epithelial barrier [32]. As an immunomodulator, L. crispatus also participates in the immunomodulation of the cervicovaginal microenvironment, which plays an important and positive role in preventing pathogen infection and even inhibiting the carcinogenesis of cervical cells [33, 34]. Although the barrier function of Lactobacilli can inhibit the entry of HPV into basal keratinocytes, not all Lactobacillus-dominant cervicovaginal microbiota is protective. Lactic acid has D- and L-isomer, while D-lactate is more effective in maintaining the vaginal homeostasis [35]. For example, D-isomers can inhibit the decomposition of extracellular matrix by down-regulating matrix metalloproteinase 8 (MMP-8), which prevents the entry of HPV into the basal keratinocytes by altering cervical integrity [36]. Moreover, it can inhibit histidine deacetylase and activate gene transcription of local innate immune molecules in vagina [36].

L. crispatus, L. jensenii and L. gasseri produce both D- and L-lactate, but L. iners and a variety of anaerobes can only synthesize L-lactate [37]. Given the quantity and stability of L. crispatus in vaginal cervicovaginal microbiota, as well as its strong ability to produce D-lactate and H₂O₂, a natural isolated L. crispatus strain was selected as the objects of our study. The anti-HPV mechanism of Lactobacillus is shown in Figure 4, indicating L. crispatus has a promising protective effect on the female reproductive tract, whose dysbiosis strongly correlates with the development of various genital tract inflammation and even HPV infection.

Through high-throughput sequencing analysis, lower levels of Lactobacilli and higher levels of Prevotella and Gardnerella has been found in patients with HR-HPV compared to that of the healthy people. After L. crispatus treatment, the abundance of Lactobacilli increased, while Prevotella and Gardnerella decreased, which tend to be similar to those of the normal healthy population. However, the mechanism by which Prevotella and Gardnerella promotes HR-HPV infection remains unclear.

Prevotella is a Gram-negative anaerobe, which is one of the dominant bacteria in human vaginal microbiota and is an opportunistic pathogen, causing diseases such as bacterial vaginosis [38]. Prevotella can produce sialidase (SNA), a group of mucin-degrading enzymes, which disrupts the integrity of mucosa as well as the epithelium to help the entry of HPV to basal keratinocytes [22, 39]. Based on transkingdom network analysis and the result of the co-culture with a cervical cancer cell line, Prevotella is found to upregulate LAMP3, a key tumor driver, which plays a crucial role in cervical cancer by driving expression of multiple antiviral genes under anaerobic conditions [40]. In addition, overgrowth of Prevotella in the vagina may influence the occurrence of persistent HR-HPV infection-related cervical lesions through host NF-κB and C-myc signaling [29].

Gardnerella is the most common microorganism identified from the vaginal samples in women with BV. It is one of the most common bacteria in the vaginal microecology of patients with HPV infection and cervical cancer [41]. It can not only form dense biofilms to adhere tightly on the surface of vaginal epithelial cells, release cytolysin and activate inflammatory responses, but also release metabolites such as producing sialidase (SNA), hemolysin to destroy vaginal epithelial cells, damage the mucosal barrier, and promoting the adhesion and invasion of pathogens, such as HPV [42, 43]. Thus, increased Gardnerella is regarded as a key co-factor of HPV infection to cervical carcinoma or precancerous lesions.

In this study, we evaluated the efficacy of intravaginal administration of L. crispatus in the treatment of patients with HR-HPV. The results showed that L. crispatus could significantly reduce the HPV viral load, improve the HPV clearance rate, improve conditions of vaginitis with no severe adverse reactions. Furthermore, L. crispatus chen-01 could effectively reconstitute the vaginal microbiota in women with HR-HPV. These results suggest that intravaginal administration of L. crispatus is a potential effective and specific treatment for patients already infected with HR-HPV. However, the underlying mechanisms of the improved clearance rate can be sophisticated, which can be achieved by the bacterial interactions, competitive adhesion or the secretion of bacterial products, etc. Therefore, exploration into the potential mechanism underlying the improved clearance rate is a further task in our following research. This treatment is simple, feasible with no obvious side effects, which has the value of clinical promotion. However, there are still limitations of this study such as the relatively short observational time, thus the long-term therapeutic effects of L. crispatus transplantation remains to be further evaluated. Moreover, difficulty in the recruitment of large samples make it harder to control the potential heterogeneity of the participants, thus hindering the current clinical research. This requires a more comprehensive study to make the further confirmation.

Materials and Methods