Table 2.

Details of papers describing detection of mucosal HPV-specific antibodies after HPV vaccination.

First author (year of publication) [ref]	Country of execution	Study Vaccine Vaccine schedule Vaccine administration	Number of participants	Mean age (total age range)	Total number of collected CVS samples (collection time (month (M)) from 1st vaccine dose)e	Methodology and antibodies analysed in CVS (class)	CVS collection method Storage	Main study outcome
1/Experimental HPV immunization
Nardelli-Haefliger et al. (2003) [55]	Switzerland	HPV16 L1 VLP vaccine different immunization/dosing schedules no adjuvant i.m.	18; (n = 7, taking oral contraceptives; n = 11, ovulating)	Not reported 18–45 y	216 (M0-M2/M5, followed by twice weekly for 5 weeks)	VLP-based ELISA anti-HPV16 L1 (IgG)	Wick Samples were placed on ice, and PBS containing protease inhibitors were added. The liquid fraction (i.e., diluted cervical secretion) was frozen at –70 °C until analysis	The cervical titers among participants in the contraceptive group were relatively constant throughout the contraceptive cycle. In contrast, the cervical titers among participants in the ovulatory group varied during the menstrual cycle
Fife et al. (2004) [50]	USA	HPV11 or 16 L1 VLP vaccine different immunization/dosing schedules i.m.	249	Median: 20 y 18–25 y	182 (M7)	VLP-based ELISA anti-HPV11/16 L1 (IgG/A)	CVL Not reported	Only about one half of the study participants who received the three highest experimental HPV11 vaccine doses had in their CVL detectable anti-HPV11 by month 7. The proportion was even lower in the HPV16 vaccine study
Nardelli-Haefliger et al. (2005) [54]	Switzerland	HPV16 L1 VLP vaccine different immunization/dosing schedules no adjuvant nasal spray/bronchial aerosol or combination i.m./aerosol	32	Not reported 18–45 y	64 (M0-M2)	VLP-based ELISA anti-HPV16 L1 (sIgA, IgG/A)	Conducted as Nardelli-Haefliger et al. (2003) [55]	Data suggest that aerosol administration of HPV VLPs may represent a potential alternative to parenteral injection. IgA was detected at the cervix in a subset of these vaccines
2/HPV-6/11/16/18 (Gardasil®) immunization
Huo et al. (2012) [52]	England	HPV-6/11/16/18 vaccine 0,1,4 month schedule Sublingual (SL) drops/i.m.	18	24.2–26.3 y 19–31 y	81 (M0-M1-M2-M4-M5)	1/VLP-based ELISA anti-HPV6/16/18 L1 (IgG/A) 2/Neutralizing assay nAb-HPV16	Wick Samples were snipped into the top chamber of a Spin-X tube containing 300  mL sterile filtered extraction buffer and centrifuged at 4 °C for 15  min at 13,000  g. A repeat extraction was performed by adding additional extraction buffer to the top chamber, and then 8  mL heat inactivated FBS added to pooled secretions from each sample site, prior to separation into 200  mL aliquots and freezing at –80 °C until analysis	SL antigens induced 38-fold lower serum and 2-fold lower cervical/vaginal IgG than i.m. delivery, and induced or boosted serum virus neutralizing antibody in only 3/12 subjects. Neither route reproducibly induced HPV-specific mucosal IgA. The observation that SL immunization could boost pre-existing serum neutralizing activity points to the possible use of i.m. prime/SL boost schedules
3/HPV-16/18 (Cervarix®) immunization
Kemp et al. (2008b) [53]	Costa Rica	HPV-16/18 vaccine 0,1,6 month schedule i.m.	50	Not reported 18–25 y	47 (M0 (n = 5) -M12)	1/Neutralizing assay nAb-HPV16/18 2/VLP-based ELISA anti-HPV16/18 L1 (IgG)	Wick Samples were placed into a 10  mL tube for storage in liquid nitrogen. After shipment the samples were stored at −70 °C. Sponge were extracted in a buffer containing PBS, NaCl and Aprotinin. 300  μl of cervical extraction buffer was slowly added to the top of the sponge. 4  μl was added FBS. The extracts were aliquoted and frozen at −70 °C until further testing	Strong correlations between SEAP-NA and ELISA were observed. Systemic and cervical antibody measures also correlated well except at mid-cycle. Correlations between antibody levels at one and twelve months following the start of vaccination were poor
Schwarz et al. (2010) [60],a	Germany, The Netherlands, Finland, USA, Poland, Denmark	HPV-16/18 vaccine 0,1,6 month schedule i.m.	350	Not reported 10–65 y	553 (M7-M12-M18-M24-M36)	VLP-based ELISA anti-HPV16/18 L1 (IgG)	Wick Conducted as Kemp et al. (2008b) [53]. Samples were stored at -20 °C or -70 °C until antibody extraction	Good correlation was seen between HPV16/18 antibody levels at all time-points. The strong correlation between levels of HPV16/18 antibodies in serum and CVS up to 36 months post-vaccination supports transudation of serum antibodies as the mechanism by which antibodies are introduced into CVS
Petäjä et al. (2011) [56]	Denmark, Estonia, Finland	HPV-16/18 vaccine 0,1,6 month schedule i.m.	321	24.2 y (Group 15–25y) 10–25 y	69 (only from the age 15–25 group; M24-M36-M48)	VLP-based ELISA anti-HPV16/18 L1 (IgG)	Wick Conducted as Schwarz et al. (2009) [61]	Anti-HPV16/18 antibodies in CVS were detectable for subjects aged 15–25 years (84% and 70%, respectively). There was a strong correlation between serum and CVS anti-HPV16/18 antibodies levels
Scherpenisse et al. (2013) [57]	The Netherlands	HPV-16/18 vaccine 0,1,6 month schedule i.m.	1,151	15.1 y 14–16 y	649 (M0-M12-M24)	VLP-based multiplex immunoassay Anti-HPV16/18/31/33 /45/52/58 L1 (IgG/A)	Wick Samples were collected from the tampons by addition of PBS containing complete protease inhibitor cocktail and subsequent centrifugation for 30  min, 3,200  g  at 4 °C. CVS samples were stored at -80 °C until analysis	Post-vaccination, HPV16/18 IgG and IgA are detectable in CVS. The correlation of HPV16/18 IgG antibody levels between serum and CVS suggests that vaccine-induced HPV antibodies transudate and/or exudate from the systemic circulation to the cervical mucosa to provide protection against HPV infections
Gonçalves et al. (2016) [51],b	Brazil	HPV-16/18 vaccine 0,1,6 month schedule i.m.	60	27.2 y 19–43 y	60 (M0-M1-M6-M7)	VLP-based ELISA anti-HPV 16/18 L1 (IgG/A)	CVL Samples were obtained by PBS (No storage temperature reported)	After the third vaccination, there is a strong agreement between cervical and systemic IgG antibody responses and a weak agreement between cervical and systemic IgA antibody responses. The induction of IgA antibodies seems to be secondary to that of IgG antibodies in response to HPV i.m. vaccination
Ferreira Costa et al. (2018) [62],b	Brazil	HPV-16/18 vaccine 0,1,6 month schedule i.m.	35	Same population as Gonçalves et al. (2016) [51]; age not specified	70 (M7-M18)	VLP-based ELISA anti-HPV/VLP (IgA/G); HPV type not specified	CVL Not reported	Cervical samples were positive for both IgG and IgA antibodies at 7 months and decreased after 1 year to 33% and 29%. The median absorbance in serum and the cervix for IgG and IgA anti-HPV-VLP antibodies was significantly higher at month 7 after vaccination when compared to 1 year post-vaccination
Schwarz et al. (2009) [47],c	Germany, Poland	HPV-16/18 vaccine 0,1,6 month schedule i.m.	531	35 y 15–55 y	149 (M18-M24)	VLP-based ELISA anti-HPV16/18 L1 (IgG)	Wick Samples were collected per woman and stored at −20 °C until antibody extraction. The extraction protocol was conducted as Castle et al. (2004) [71]	There was a high correlation between HPV16 and HPV18 antibody levels (IgG) in CVS and sera, regardless of age
Schwarz et al. (2015) [58],c			488	Not reported 15–55 y	190 (M60-M72)			A strong correlation between anti-HPV16/18 levels in serum and CVS samples 6 years after vaccination indicates a long-lasting transudation of serum antibodies across the cervical epithelium
Schwarz et al. (2017)[59],c			470	Not reported 15–55 y	107 (M120)			Correlation coefficients for antibody titers in serum and CVS were 0.64 (anti-HPV16) and 0.38 (anti-HPV18)
4/HPV-16/18 (Cervarix®) vs HPV-6/11/16/18 (Gardasil®) immunization
Draper et al. (2013) [46]	UK	HPV-16/18 vaccine HPV-6/11/16/18 vaccine Both 0,1,6 month schedule i.m.	198	Not reported 12–15 y	50 (M7)	VLP-based ELISA anti- HPV16/18/31/45 L1 (IgG)	Cytobrush Samples were rehydrated with PBS and subjected to centrifugation within a Amicon tube for 5  min  at 2,500  g. Two such extractions were performed and the eluted material pooled and subjected to centrifugation at 13,000  g to remove cellular debris. The clarified supernatant was aliquoted and stored at -80 °C	Levels of neutralizing and binding antibodies in genital secretions were closely associated with those found in the serum, with Cervarix® having a median 2.5 fold higher level of HPV-specific IgG ratio in serum and genital samples than Gardasil®
Einstein et al. (2009) [47],d	USA	HPV-16/18 vaccine 0,1,6 month schedule i.m. HPV-6/11/16/18 vaccine 0,2,6 month schedule i.m.	920	30.2-30.7 y 18–45 y	165 (M0-M7)	1/Neutralizing assay nAb-HPV16/18 2/VLP-based ELISA anti-HPV16/18 L1 (IgG)	Wick Conducted as Schwarz (2009) [61]	Positivity rates for anti-HPV16/18 nAb in CVS and circulating HPV16/18-specific memory B-cell frequencies were higher after vaccination with Cervarix® compared with Gardasil®
Einstein et al. (2011) [48],d			799	31 y 18–45 y	222 (M12-M18-M24)	VLP-based ELISA anti-HPV16/18 L1 (IgG)		Positivity rates and levels of antigen-specific IgG antibodies in CVS were not significantly different between vaccines
Einstein et al. (2014) [49] d			524	31 y 18–45 y	170 (M36-M48)			Limited CVS samples were available. Positivity rates of anti-HPV16/18 IgG antibodies in CVS appeared higher in the Cervarix® group compared with the Gardasil® group, while CVS antibody levels were of similar level. Antibody levels in serum and CVS are poorly correlated, especially for HPV18
Barr, Koutsky (2004) [45]	Letter to Nardelli-Haefliger et al. (2003) [55]

^aThis pooled analysis from four separate phase III clinical trials includes data from Einstein et al. (2009) [47] and Schwarz et al. (2009) [61].

^bFerreira Costa et al. (2018) is a follow-up study of Gonçalves et al.(2016) reporting 7–18 months after HPV-16/18 vaccination.

^cSchwarz et al. (2009/2012/2017) is a follow-up study with reports 24-72-120 months after HPV-16/18 vaccination.^d Einstein et al. (2009/2011/2014) is a follow-up study comparing HPV-16/18 and -6/11/16/18 vaccination after 7-24-48 months.

^dSeveral CVS samples were still excluded because of weight, blood contamination, etc. Abbreviation: VLP, virus-like-particles; i.m., intramuscular; OC, oral contraceptives; nAb, neutralizing antibodies; FBS, Fetal bovine serum; PBS, Phosphate buffered saline; SEAP-NA, secreted alkaline phosphatase neutralization assay.