E. coli through N-terminal truncation of HPV L1 proteins and non-fusion soluble expression. Second, based on genetic relationships and an L1 homologous loop-swapping rationale, we constructed several triple-type chimeric VLPs for HPV39, −68 and −70, and obtained the lead candidate named H39–68FG-70DE by the immunogenicity optimization using reactivity profile of a panel type-specific monoclonal antibodies. Through comprehensive characterization using various biochemical, VLP-based analyses and immune assays, we show that H39–68FG-70DE assumes similar particulate properties as that of its parental VLPs, along with comparable neutralization immunogenicity for all three HPV types. Overall, this study shows the promise and translatability of an HPV39/68/70 triple-type vaccine, and the possibility of expanding the type-coverage of current HPV vaccines. Our study further expanded the essential criteria on the rational design of a cross-type vaccine, i.e. separate sites with inter-type similar sequence and structure as well as type-specific immunodominant epitope to be clustered together.In vaccinology, a potent immunogen has two prerequisite attributes—antigenicity and immunogenicity. We have rational designed a triple-type HPV vaccine against HPV58, −33 and −52 covered in Gardasil 9 based on the sequence homology and similar surface loop structure of L1 protein, which is related to cross-type antigenicity. Here, we design another triple-type vaccine against non-vaccine types HPV39, −68 and −70 by immunogenicity optimization considering type specific immunodominant epitopes located in separate region for different types. First, we optimized the expression of wild-type HPV39, −68 and −70 L1-only virus-like particles (VLPs) in  Indeed, according to the World Health Organization (WHO), in 2020, ~ 604,127 women were newly diagnosed with cervical cancer worldwide, with ~341,831 deaths. Studies have shown that the main cause of cervical cancer and other genital cancers is persistent infection with high-risk human papillomavirus (HR-HPV)6. To date, over 450 distinct HPV types have been identified and classified into 4 groups by the WHO International Agency for Research on Cancer (IARC) working group according to their ability to generate malignancies9: class 1 (carcinogenic) includes HPV16, −18, −31, −33, −35, −39, −45, −51, −52, −56, −58 and −59; class 2 A (probably carcinogenic) includes HPV68; class 2B (possibly carcinogenic) includes HPV26, −30, −34, −53, −66, −67, −69, −70, −73, −82, −85 and −97; class 3 (not classifiable) includes HPV6 and −11, and class 4 is considered probably non-carcinogenic to humans11. Class 1 and 2 HPV types are highly correlated with cervical cancer and are classified as high-risk (HR-HPV), whereas class 3 types, HPV6 and −11, are mainly associated with genital warts and are classified as low-risk types (LR-HPV)12.Cervical cancer has become the fourth-most prevalent cancer in women and poses a serious threat to women’s health14. Despite the broad spectrum of protection offered by these marketed vaccines, approximately 10% of cervical cancer caused by other HR-HPV types still cannot be effectively prevented15. Furthermore, with time and with the increasing popularity of vaccine administration against HPV, the prevalence of the non-vaccine types showed increasing to some extent in several types. In a study, these non-vaccine HPV types  were found to have increased prevailing ratio in some regions where Gardasil were extensively used for ten years16. A recent meta-analysis by Mesher et al. also showed an increase in HPV39, −52, −53 and −73 in the post-Gardasil vaccine era, and among those 20–24 years of age, evidence indicated increased prevalence of HPV7017. Consequently, there is a demand for vaccines with broader prevention efficacy.Five HPV vaccines have been licensed to date: three bivalent vaccines , a quadrivalent vaccine (Gardasil [Merck & Co.]) and a nonavalent vaccine (Gardasil 9 [Merck & Co.]). Of these, Gardasil 9 offers the most protection, and is effective against ~90% of cervical cancer induced by 7 types of HR-HPV  as well as against 90% of genital warts induced by LR-HPV types (HPV6 and −11)11. HPV39 ranks ninth among the ten most frequent HPV oncogenic types in Chinese women with cervical cancer, and in southern parts of China, HPV39 prevails just after HPV52, −16, −58 and −1818. In other regions, the prevalence of HPV39 is similar, ranking eighth frequent HPV carcinogenic types in developed countries, and ranking tenth in developing regions. HPV68 and −70 also prevail globally after HPV 39, despite of relatively lower frequency11. Given the HPV39, −68 and −70 prevailing while not being protected by Gardasil 9, a next-generation of HPV vaccine should have broader coverage and preferably includes these three carcinogenic HPV types.According to the WHO report, the global prevalence of HR-HPV39, −68 and −70 accounted for 1.5%, 0.8% and 0.2% respectively in the cases of cervical cancer in 202021. The five marketed vaccines are all based on L1-only VLPs23:Cervarix25 Cecolin27, and a HPV16/18 bivalent vaccine produced in Pichia pastoris by Walvax Biotechnology Co., Ltd. contain VLPs for HPV16 and −18; Gardasil comprises VLPs for HPV6, −11, −16 and −1829, with Gardasil 9 adding in VLPs for HPV31, −33, −45, −52 and −5814. Each vaccine is formulated through a mixture of VLP types meaning that they are essentially type-specific and offer limited broad-spectrum protection. One could predict that including additional HPV VLPs into the vaccine production strategy could broaden the protection rate; however, studies show that as the number of VLP types increases, the protein content in the vaccine also increases substantially, which, in turn, requires the administration of a much higher dose of vaccine30. Already, one dose of Gardasil 9 contains up to 270 µg of total HPV L1 proteins. For humans, the injection of an excessively high dosage of a foreign protein can induce significant side effects. In addition to dosage considerations, adding more HPV types will increase the complexity of the vaccine production process, and may generate immune competition among the different VLPs31. Increasing the number of VLP types is therefore considered a very challenging approach.As a non-enveloped, double-stranded DNA virus, the HPV capsid is composed of the major structural protein, L1, and the minor structural protein, L2. L1 proteins can self-assemble into virus-like particles (VLPs) and induce a strong immune response34. Recent innovation in structural vaccinology suggest that protein structural information can be used to optimize and achieve precise antigen functional epitopes that can be harnessed for immunogen design. Indeed, studies show that the effective presentation and/or integration of functional epitopes can trick the innate immune spectrum into producing more complex, broad-spectrum responses35. One example is relatively conserved immunodominant epitopes were grafted onto viral surface proteins using a germline-targeting strategy in the development of universal HIV vaccines36. Another method widely used in vaccine design is computationally optimized broadly reactive antigen method, or COBRA37. The principle in this method is to construct a consensus sequence from multiple immunodominant sequences from different strains to achieve cross-type protection38. For influenza viruses, a recombinant hemagglutinin (HA) polypeptide constructed by fusing the sequences from H5N1 and H1N1 can be used to elicit a broadly reactive immune response against both H5N1 and H1N1 influenza strains (patent no. EP2616545A4). Another vaccine, Human COBRA 2, designed by incorporating key neutralizing epitopes of two HA antigens-Human COBRA 2 (Hu-CO) and Human-Avian COBRA 2 (Hu-Av CO)-also elicits broadly protective antibodies against heterologous clades of viruses39.Nearly all of the marketed vaccines offer protection by inducing neutralizing antibodies; however, for highly antigenically variable pathogens, such as HPV, HIV and influenza, traditional vaccine strategies provide limited broad-spectrum protection against these variations40, whereas most of neutralizing antibodies recognize those conformational epitopes that comprise some of the five surface loops42. For instance, the well-known HPV16.V5 is an HPV16-specific neutralizing antibody, which showed immune predominant frequency in the sera of native HPV-infected humans and is of clinical importance43. HPV16.V5 was identified to recognize the FG loop (aa266~297) and HI loop (aa339~365) of L1 protein, and could neutralize virus infection by interfering with the endocytosis of virus entry44. The immune complex structure of HPV16.U4, HPV16.14J and HPV16.1A with HPV16 VLP also showed that the neutralizing epitope of HPV L1 was located at the five surface loop regions 46. Surface loops of genetically close HPV types are known to have a highly similar conformation47. In previous studies, we designed a chimeric VLP of the genetically close HPV58/33/52 based on sequence homology of L1 proteins and structural information of the surface loops, which elicited high levels of cross-neutralizing antibodies in both mice and non-human primates48. However, why single chimera VLP could work well for producing cross-neutralization against three HPV types, and which loop could be an ideal target for clustering remains to be determined. Here, we demonstrate the potent elicitation of the neutralizing antibody depends not only the resurface epitope success, but also its immunodominant nature for producing antibody, and the potential universality of this design through the characterization of a single-particle cross-type vaccine candidate that can simultaneously prevent carcinogenic types HPV39, −68 and −70. Using type-specific monoclonal antibodies and pseudovirus platforms, we analyzed the antigenicity and immunogenicity of this cross-type candidate vaccine, and confirmed that the HPV loop transplantation strategy is feasible between phylo-genetically close HPV types. Remarkably, genetically close types means the types belong to a same phylogenetic group, whereas genetically distant types fall in different group in the phylogenetical tree of HPV by the alignment of L1 amino acid sequences48. Dominant loop transfer provides a new level of antigenicity and immunogenicity for the chimeric VLP without destroying the backbone types, thus achieving cross-type protection with fewer immunogens. This chimeric VLP also fills the present gap in HPV prevention against HPV39/68/70.Both linear and conformational epitopes have been identified on the surface of HPV L1 VLPs11, Supplementary Fig. Using data from the 2021 WHO epidemiological statistics reportWe divided the 27 HPV types into 8 phylo-genetically close groups according to the evolutionary distance of the L1 protein. As shown in Supplementary Fig. E. coli system. Full-length and a series of N-terminal truncations  of HPV39, −68 and −70 L1 proteins were constructed to optimize expression. Soluble and highly expressed constructs with a purity of >95%—HPV39 (15–505 aa), HPV68 (0–505 aa) and HPV70 (10–506 aa)—were purified by two-step chromatography. The molecular weights were determined to be ~55 kD for all three constructs , 1280 to 10,240  and 1280 to 10240 ; these were all significantly higher than those elicited by the WT HPV39 VLPs (anti-HPV68 titers of 2.5 to 80). Similarly, H68-39BC and H68-39HI elicited high neutralizing titers against HPV39  as compared with WT HPV68 VLPs (anti-HPV39 titers of 2.5–400).We then further assessed the immunogenicity of these chimeric HPV39/68 VLPs. BALB/c mice were inoculated by intraperitoneal injection three times with chimeric VLPs formulated with aluminum adjuvant. The neutralization titers of the sera after three rounds of immunization were measured with a pseudovirus-based neutralization assay (PBNA). We found a dose-dependent immunization effect for both WT VLPs and HPV39/68 chimeric VLPs Fig. , with hi50); i.e., the minimum effective dose at which 50% of the experimental animals seroconverted in a given group53. As shown in Table 50 values for WT HPV39 and HPV68 were 0.022 μg and 0.023 μg, respectively. The chimeric VLPs similarly provided neutralization activity; albeit, with differing levels. For H39–68FG, the ED50 value for HPV39 was 0.091 μg; for H68-39BC and H68-39HI, the ED50 values for HPV68 were 0.021 μg and 0.028 μg, respectively; these results indicate that these chimeras offered neutralization activity against the backbone type of the chimera to levels similar to that of the WT HPV39 or HPV68. In contrast, H39–68FG provided lower ED50 against HPV68 (ED50 = 0.405 μg) than HPV39 against HPV 68 (>0.900 μg), and H68-39BC provided lower ED50 against HPV39 (ED50 = 0.395 μg) than HPV68 against HPV 39(>0.900 μg), suggesting that a single surface loop grafted onto a heterologous backbone type also has the ability to induce neutralizing antibodies against the donor loop type. Thus, the chimeric VLPs, H39–68FG and H68-39BC, with “one anti-two” cross-neutralization activity, could be used as vaccine antigens for the generation of vaccines.H39–68FG, H68-39BC and H68-39HI all exhibited good double-type cross-neutralization potency. Therefore, we next subjected these chimeras to further study to determine the half-effective dose , compared with WT HPV39 , 0.030 μg (anti-HPV68) and 0.040 μg (anti-HPV70). H39–68FG-70EF and H39–68FG-70HI also showed good triple-type cross-neutralization and could prevent HPV39, −68 and −70 infections. Overall, H39–68FG-70DE and H39–68FG-70HI were chosen as the best cross-type vaccine candidates for further study.We then went on to measure the EDWe next sought to assess batch consistency, and confirmed that both of the cross-type vaccine candidates, H39–68FG-70DE and H39–68FG-70HI, were produced with excellent quality and consistency of purity, particle morphology, and homogeneity among batches Fig. . We also50) of binding via ELISA  against WT HPV39/68/70 VLPs. This mAb panel was then used to determine the median effective concentration . As shown in Fig. 54. As well-known in vaccinology, there are two prerequisite attributes for a potent immunogen, i.e. antigenicity based on specific epitope structure and immunogenicity raised from those immunodominant epitopes. The effectiveness of most vaccines depends on nAb response; however, most protective nAbs are type-specific. Therefore, for pathogens that frequently mutate or with multiple genotypes, such as HPV, it is becoming increasingly important to determine new ways to optimize neutralizing epitopes while achieving broad immune protection. At present, there are several immunogen design strategies in use for the development of broad-spectrum vaccines, such as germline targeting, COBRA, consensus sequence construction, and structural biology-based epitope transplantation. For instance, structural analysis of multiple variants of the factor H binding protein (fHbp) on the surface of Neisseria meningitidis (MenB) led to the identification of key functional epitopes on different variants, and the successful design of a new epitope through transplantation, offering broad protection against group B meningitis infection56. These methods could be explored in the development of HPV vaccines against multiple types of viruses.Rational immunogen design is expected to solve the current challenges facing vaccine developmentIn our previous study, we designed a triple-type HPV chimera based on higher sequence homology of three types of L1 proteins and similar structures of the five surface loops. The chimera VLP is derived from a basic type of HPV VLP (HPV58 as example) with two surface loops swapped by HPV33 BC and HPV52 HI, which have similar loop conformation and fewer sequence discrepancy between each other types. We established the proof-of-concept of the relational design of triple-type HPV vaccine. However, we did not address why single chimera VLP could work well for producing cross-neutralization against three HPV types, and which loop could be an ideal target for clustering remains to be determined, which is beyond the structural information, because the potent elicitation of the neutralizing antibody depends not only the resurface epitope success, but also its immunodominant nature for producing antibody. In this study, we showcase another chimera HPV39/68/70 by further optimization in the second attribute, i.e. the immunogenicity related to type specific immunodominant epitopes located in separate region for different types.50 values [<50 ng/mL] with H39–68FG-70DE rather than that of H39–68FG-70HI), the region covering DE loop might be more immunogenic than HI loop in HPV70 L1 and therefore enables the H39–68FG-70DE to elicit higher and balanced triple-type neutralizing antibody response both in ED50 and antibody titration assays. Thus, we further underscore the immunological consideration-separate immunodominant site for various types or pathogens should be clustered-on chimera design that is usually based on sequence and structural information. Overall, we show that by simply translocating just 16 amino acids, we could successfully construct a triple-type chimeric VLP that can induce cross-neutralization against HPV 39/68/70 at the same time.Here, we engineered a single VLP immunogen that contained the immunodominant regions of three HPV genotypes, focusing on those genotypes not already covered or presumably only partly covered by existing vaccines. Based on a consensus sequence strategy, we successfully integrated the immunodominant epitopes of HPV types 39, 68 and 70 through epitope transplantation, and elicited protective antibodies against all the three serotypes. We sought to explain the reasons for the successful of this epitope exchange strategy from two aspects of antigenicity and immunogenicity. First, in comparing the immunogenicity of WT and chimeric VLPs, we identified that all five surface loops played a role in eliciting nAbs. Through our analysis, we show that the immunodominant region of each HPV type is distinct, and that alternative immunogenic epitopes of variants 2 or 3 could be grafted onto the backbone of variant 1, offering cross-neutralization. We show that the FG loop is the dominant neutralizing epitope of HPV 68, when we then swapped the FG loop of HPV68 into HPV39, the chimeric H39–68FG elicited a neutralizing response against HPV68, with high antibody titers. Comparatively, we find that the DE and HI loops of HPV70 elicit nAbs against HPV70, and integrated these loops into the double-type chimera. In terms of antigenicity, H39–68FG-70DE shows overall higher reactivity with HPV70 specific mAbs than H39–68FG-70HI  regulation. On the other hand, the chimera VLP actually cluster major neutralization sites of three HPV types into single VLP and might diminish the non-neutralization antibody elicitation than three WT VLPs as immunogen. Moreover, based on the evolutionary relationship, the progressive chimeric antigen screening strategy development in this study has greatly improved the screening efficiency of candidate vaccines. Theoretically, there are at least 60 triple-type chimeric VLP combinations achievable through surface loop swapping. Yet, using the strategy presented in this study—preferentially screening double-type chimeric VLPs with a closer genetic relationship and then adding in the third—reduced the number of chimeras that needed to be verified. Considering that at least 25 HPV types are highly related to cervical cancer, if the antigen-antibody interaction sites can be precisely located, a wider spectrum of protection can be achieved by transplanting fewer amino acids and fusing more types of immunodominant epitopes.In summary, we verified the feasibility and effectiveness of homologous loop-swapping as a strategy to induce cross-neutralization among different but closely related HPV types, and obtained a vaccine molecule H39–68FG-70DE that can achieve triple-type cross-neutralization to prevent infection from high-risk carcinogenic HPV39/68/70. The finding and analysis about the chimera of HPV39, −68 and −70 in this study could further expand the knowledge and consideration on HPV chimera vaccine design from the immunological aspect, going beyond the sequence and structural information. Future clinical work will ascertain the feasibility and commercial utility of this H39–68FG-70DE chimera.The experimental protocols were approved by the Xiamen University Laboratory Animal Management Ethics Committee. All manipulations were strictly conducted in compliance with animal ethics guidelines and approved protocols.E. coli strain for L1 protein expression.N-terminally truncated HPV39 (P24838), HPV68 (AAZ39498.1) and HPV70 (P50793) L1 genes were cloned into the pTO-T7 expression vector by Gibson assembly, and plasmids were transferred into the ER2566 E. coli strains containing HPV39, −68, −70 WT or the chimeric L1 plasmid were cultured in LB liquid medium at 37 °C until reaching an OD600 of 0.6. Protein expression was then induced by the addition of isopropyl-β-D-thiogalactoside  and samples were further cultured at 24 °C for 10 h. Cells were then harvested by centrifugation , resuspended in lysis buffer  and lysed by ultrasonication. The ultrasonication output power was set at 45% of 500 W, and the ultrasonication time was 15 min per gram of E. coli, with a frequency of 2 s pulsed and 4 s paused. HPV L1 proteins were released from the cells and treated with 20 mM DTT for 12 h at room temperature. Then the supernatant was purified by chromatography using SP or XS sepharose . For the preparation of VLPs, the purified L1 protein was then dialyzed into a neutral buffer  without DTT to allow VLP self-assembly and purified by Superdex 200.ER2566 P24838), HPV68 (AAZ39498.1), HPV70 (P50793), HPV6 (7EW5_A), HPV30 (YP_009508159.1), HPV34 (NP_041812.1), HPV51 (ACV88631.1), HPV58 (P26535.1), HPV66 (ABO76865.1), HPV67 (ALT54969.1), HPV69 (AHV83654.1), HPV73 (ALJ32570.1), HPV82 (ALJ32344.1), HPV85 (YP_009362313.1), HPV97 (ABO27083.1), and HPV11, −16, −18, −26, −31, −33, −35, −45, −52, −53, −56, −5951 L1 genes were from NCBI. L1 protein sequence alignment was performed using BLAST, and evolutionary tree building was performed by the Neighbor-joining method in MEGA X software (https://www.megasoftware.net).HPV39  SDS, 20% (w/v) glycerol, 200 mM dithiothreitol, and 0.002% (w/v) bromophenol blue) to a final protein concentration of 0.3–0.5 mg/mL, heated at 80 °C for 10 min, and then loaded into the wells of a 10% separating gel. The gels were subjected to standard laboratory methods for gel electrophoresis  and stained with Coomassie Brilliant Blue R-250 (Bio-Rad) for 30 min at room temperature.For western blotting, gels were transferred to nitrocellulose membranes, blocked with 5% skimmed milk for 1 h, and then incubated with HPV broad-spectrum linear monoclonal antibody 4B3 (1:1000 dilution) for 1 h at room temperature. Membranes were washed, and then incubated with goat anti-mouse alkaline phosphatase-conjugated antibodies  for 1 h. NBT/BCIP  reagent was used to develop the color (5 min). All blots or gels derive from the same experiment and that they were processed in parallel. And source data are provided in Supplementary Fig. Samples were diluted to 0.15 mg/mL with HPV L1 storage buffer. Each sample (10 μL) was pipetted onto carbon-coated copper grids for 10 min, absorbing any residual liquid before adding dropwise 2% phosphotungstic acid (pH 6.4) and incubating the reaction for 5 min. An FEI Tecnai T12 TEM with an accelerating voltage of 120 kV was used to observe and measure HPV VLP morphology.The homogeneity of WT and chimeric VLPs was assessed using an Agilent 1200 high-performance liquid chromatography system, with a pre-installed TSK G5000 pwxl 7.5 mm × 300 mm column . The system flow rate was 0.5 mL/min, and the detection wavelength was set to 280 nm.Sedimentation analysis was carried out on a Beckman XL-A analytical ultracentrifuge at 20 °C. 10 mM phosphate buffer pH 6.5 with 0.5 M NaCl was used as the reference solution. WT and chimeric VLPs were diluted to 0.5 mg/mL with reference solution, and centrifuged at 3951 × g using an An-60 Ti rotor . The sedimentation coefficient was determined using Sedfit software kindly provided by Dr. P. Schuck at the National Institutes of Health .The thermal stability of WT and chimeric VLPs was measured using MicroCal VP-Capillary differential scanning calorimetry (DSC) . Samples were diluted to 0.5 mg/mL with 10 mM phosphate buffer, pH 6.5, with 0.5 M NaCl. The temperature range was set as 10 °C to 90 °C, with a scanning rate of 100 °C/h. MicroCal Origin 8.0 software  was used to analyze the DSC curve and the melting temperatures (Tm) of the samples.2SO4. An automated ELISA reader  was used to detect the absorbance at 450 nm . GraphPad Prism  were used to calculate the median effective concentration , which is the concentration of antibody that binds to 50% of the antigen.The wells of 96-well microplates were coated with WT or chimeric VLPs (300 ng per well) for 2 h at 37 °C and then blocked with 200 μL blocking solution for 2 h at 37 °C. The wells were then incubated with 100 μL of twofold serially diluted mAbs at a starting concentration of 1 μg/mL for 45 min at 37 °C. The wells were then washed and incubated with 100 μL horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody , followed by 50 μL of 3, 3’, 5, 5’-tetramethylbenzidine liquid substrate  per well for 10 min at 37 °C and the reaction was quenched with the addition of 50 µL 2 M H49 or cisTEM50 software. The resolutions of the final 3D density maps were estimated based on the gold-standard FSC curve with a cut-off of 0.14357. Visualization of the density maps were performed with software ChimeraX58.The WT and chimeric VLPs (~2.0 mg/mL) were vitrified on Quantifoil holey carbon grids using an automated Thermo Fisher Vitrobot IV, and the cryo-EM images were collected using the Thermo Fisher TF30 electron microscope equipped with Thermo Fisher Falcon II and Falcon III cameras. The acceleration voltage was set to 300 kV and the magnification was set to 93,000×, resulting in a pixel size of 1.120 Å. Data were collected automatically by using Thermo Fisher EPU software. Icosahedral 3D reconstruction were performed by using AUTO3DEMHPV WT and chimeric VLPs, formulated with Freund’s complete or incomplete adjuvant (20 μg/dose), were subcutaneously injected into BALB/c mice, with three injections delivered in 2-week intervals. The first injection used Freund’s incomplete adjuvant whereas the last two injections used Freund’s complete adjuvant. Cell fusion experiments were performed 2 weeks after a boost immunization. Fused hybridomas were isolated through hypoxanthine-aminopterin-thymidine medium  selection, and supernatants were screened by a indirect ELISA and PBNA for reactivity. Positive cells were cloned by limiting dilution at least three times until a single cell clone was attained.g for 10 min, and the pellet was resuspended with 0.2 M Na2HPO4, and finally purified by protein A affinity chromatography. The resultant purified anti-HPV39 mAbs, anti-HPV68 mAbs and anti-HPV70 mAbs were diluted to 1.0 mg/mL in PBS and stored at −20 °C.The screened monoclonal hybridoma cells were injected into the peritoneal cavities of pristane-primed BALB/c mice. Ascitic fluid was collected after 9–12 days, and added with an equal volume of saturated ammonium sulfate solution, incubated at 4 °C for 30 min, centrifuged at 13,000 × 59. HPV39, -68 and -70 pseudoviruses (PsVs) were produced in HEK293FT cells, as follows. First, HEK293FT adherent cells were prepared with high activity and passaged one day before plasmid transfection, and when the cell density reached to 80–90%, then half of the medium was changed to ES serum-free dulbecco’s modified eagle medium (DMEM), the cell culture was incubated at 37 °C for 30 min. Second, the transfection system with 0.15 M NaCl buffer were prepared, 1 mL of the transfection system contains 10 μg of co-expressing plasmid carrying codon-optimized HPV L1-L2, 10 μg of pcDNA3.1-EGFP plasmid, and 65 μg of PEI (Yeasen Biotechnology (Shanghai) Co. Ltd.). The system was mixed and incubated at room temperature for 15 min, protected from light throughout. Third, the plasmid and PEI mixture were slowly added to the cells, 1 mL per plate, incubated at 37 °C for 4 h, then half of the medium was changed to ES serum-containing DMEM, and 0.5 mL ES serum was added to each cells plate, cultivated at 37 °C for 72 h. Fourth, the medium was aspirated and the cells were carefully washed with 2 mL DPBS, then resuspended with 4 mL DPBS and collected by centrifugation at 1,300 × g for 3 min at room temperature. Fifth, the cells were then lysed in 150 μL cell lysis buffer comprising 0.5% Brij58 (Sigma-Aldrich), 0.2% Benzonase , 0.2% Plasmid-Safe ATP-Dependent DNase  and DPBS-Mg solution for 24 h at 37 °C. Finally, the above cell lysate was ice-bathed for 15 min, and 5 M NaCl of 0.19 times the volume of the lysate was added, ice-bathed for 15 min too after mixing, and then centrifuged at 1,300 × g for 10 min at 4 °C, the obtained supernatant was the pseudovirus used in neutralization assay.The pvitro (for HPV 68 and 70) or pshell (for HPV39)-L1-L2 co-expression vector and the pcDNA3.1-EGFP expression vector used in the experiment were kindly provided by Dr. J. T. Schiller50) of the supernatant was then measured to determine the titers of the PsVs, calculated according to the classical Reed–Muench method60. First, HEK293FT cells in seeded into the wells of a 96-well plate at a density of 1.5 × 104 cells/well and cultured for 4–6 h at 37 °C, and then a 10-fold gradient dilution was performed on the pseudovirus, with a total of 7 gradients. Second, the pseudovirus were added to the cells plate at the ratio of 100 μL/well, 8 wells of each gradient were repeated, cultured at 37 °C for 72 h. Third, the expression of EGFP in each well of each gradient were recorded by an inverted fluorescence microscope, and the proportional distance was calculated according to the Reed-Muench method: proportional distance = (infection rate at dilution next above 50%-50%) / (infection rate next above 50% - infection rate next below 50%); and the logarithm TCID50 can be obtained by multiplying the logarithm of the dilution coefficient the proportional distance, and plus the highest dilution of the virus with an infection rate higher than 50%. So the TCID50 value can be obtained too, and PsVs were diluted to 3 ×105 TCID50/μL for PBNA assay.The tissue culture infectious dose (TCID4 cells/well and cultured for 4–6 h at 37 °C. Antibody sera were first diluted according to different dilution (2.5–1000) with ES serum-containing DMEM, and then subjected to 2-fold serial dilutions, for a total of 12 gradients, and PsVs were diluted with ES serum-containing DMEM to 3 × 105 TCID50/μL. Sera and PsVs were then mixed at a ratio of 1:1 (60 μL each), and incubated at 37 °C for 1 h. Equal volumes (60 μL) PsV diluent and culture medium mixture was used as the negative control. Cells were then incubated with 100 μL of PsV-serum mixture for 72 h at 37 °C. The number of green fluorescent spots in each cell well was read with Elispot, and the PsV infection of the cell wells with the spots number lower than half of the average value of the sum of the negative control wells were considered to inhibited by serum neutralizing antibodies. Then the neutralization titers of the antibodies were calculated as the log10 of the highest sera dilution with a percentile of infection inhibition higher than 50%.HEK293FT adherent cells were prepared with high activity and passaged one day before PBNA assay, and when the cell density reached to 80–90%, seeded into the wells of a 96-well plate at a density of 1.5 × 10n = 5) were immunized intraperitoneally three times at an interval of 2 weeks  with WT or chimeric VLPs diluted in aluminum adjuvant prepared at three different doses . In the mixed WT VLP group, the dose of each VLP was equal to that of the same VLP in the single-type groups. Serum samples were collected at week 8, and the neutralizing titers were analyzed by PBNA.To initially assess the immunogenicity of chimeric VLPs, special pathogen-free (SPF) BALB/c female mice  were vaccinated via an intraperitoneal injection of a single dose  of WT or chimeric VLPs diluted in aluminum adjuvant. In the mixed WT VLP group, the dose of each VLP was equal to that of the same VLP in the single-type groups. Serum samples were taken 5 weeks after immunization, and the ED50, , were analyzed based on a dose-response curve using the Reed–Muench model60.For EDP values < 0.05 was considered statistically significant.SPSS statistics  was used for statistical analysis of data sets. All data were analyzed by one-way analysis of variance (ANOVA) and are presented as the mean ± standard deviation (SD). Further information on research design is available in the NPJVACCINES-01815R2_Supplementary information_all_clean__Oct-10thDataset 1Reporting Summary


Intellectual humility, which entails openness to other views and a willingness to listen and engage with them, is crucial for facilitating civil dialogue and progress in debate between opposing sides. In the present research, we tested whether intellectual humility can be reliably detected in discourse and experimentally increased by a prior self-affirmation task. Three hundred and three participants took part in 116 audio- and video-recorded group discussions. Blind to condition, linguists coded participants' discourse to create an intellectual humility score. As expected, the self-affirmation task increased the coded intellectual humility, as well as participants’ self-rated prosocial affect (e.g. empathy). Unexpectedly, the effect on prosocial affect did not mediate the link between experimental condition and intellectual humility in debate. Self-reported intellectual humility and other personality variables were uncorrelated with expert-coded intellectual humility. Implications of these findings for understanding the social psychological mechanisms underpinning intellectual humility are considered. UnfortuAn antidote to this situation, intellectual humility, is receiving increasing attention. Philosophers understand intellectual humility as the disposition to own or accept one's intellectual shortcomings out of a genuine desire for knowledge and truth ,4. This et al. . In addition, all 18 emotion items and the prosocial emotions combined were uncorrelated with the behavioural measure of intellectual humility, rs ≤ 0.10. Thus, we obtained no evidence that prosocial emotion mediates the impact of the self-affirmation intervention on intellectual humility in debate.We tested whether the effect of self-affirmation on the behavioural measure of intellectual humility was dependent on the effect on prosocial emotion. We conducted a mediation analysis with condition as predictor, the aggregate index of prosocial emotion as the mediator, and the measure of intellectual humility in debate as the outcome. The bootstrapped , we coded intellectual humility from dialogue in face-to-face debates on a contentious issue and tested whether an intervention previously used to decrease psychological defensiveness increased intellectual humility in this context. Results indicated that the intervention, which affirmed the self-concept by inducing participants to think about their most important values, subsequently increased intellectual humility. Consistent with Crocker et al. , this seOther aspects of the findings were surprising. First, the effect of self-affirmation on intellectual humility was not mediated by its impact on prosocial emotion—these effects were independent. This lack of mediation might be consistent with the larger pattern of effects on emotions reported by participants. In addition to feeling more prosocial emotion after self-affirmation, they also reported feeling more pride, vulnerable, sad, selfish, scared and less content. These latter findings may reflect a more complex effect of self-affirmation on emotions, perhaps as a result of social interconnectedness activated in the task, such as pride in one's connections alongside the recognition of self–other conflicts in this interconnectedness (e.g. selfish and vulnerable).per se. It might be the case that a more focused examination of felt threat and anxiety in future research would reveal support for the hypothesis that self-affirmation builds resources that attenuate the impact of these emotions. The literature on self-affirmation effects also points to an increased sense of self-integrity )lination , which mdifferential responding to the strong and weak arguments included in the debates. In other words, it did not cause consistently more agreement with the stronger arguments and consistently less agreement with the weaker arguments. If this effect had occurred, it would have extended prior evidence of greater open-minded scrutiny of arguments presented in a videotape containing strong and weak arguments following self-affirmation [et al.'s participants (those who considered the target issue to be highly important to them), we recognized this possible impact of self-affirmation beforehand and therefore tested it. However, we regarded it as a very weak possibility because of a crucial difference between the past research and the design of our experiment. Specifically, Correll et al.'s participants did not discuss the arguments directly in their own debate. Our introduction of this social dialogue about the arguments forced high scrutiny of the arguments for participants in both experimental conditions, and this message processing constraint has been shown to eliminate argument strength effects [The second finding of note is that it is interesting that self-affirmation did not cause irmation . Althoug effects . Consequhttp://discovervalues.org/). Such public engagement events are great places to run scientific studies . They eIn sum, our research demonstrates that intellectual humility in a debate can be enhanced through a relatively simple intervention. Put numerically, 60.6% of participants in the affirmation condition showed more intellectual humility in debate than the average person in the control condition. This finding, and to some extent the concomitant effect on prosocial emotion, reveal promising implications for improving the quality of dialogue on controversial issues. We hope future research follows up on these implications. Although the study of intellectual humility is difficult due to complexities in its conceptualization and measurement, any progress in understanding its antecedents has important potential to aid democratic processes in addressing the pivotal issues of our time.


The purpose of this study is to achieve a novel and efficient method for treating the interval coefficient linear programming (ICLP) problems. The problem is used for modeling an uncertain environment that represents most real-life problems. Moreover, the optimal solution of the model represents a decision under uncertainty that has a risk of selecting the correct optimal solution that satisfies the optimality and the feasibility conditions. Therefore, a proposed algorithm is suggested for treating the ICLP problems depending on novel measures such as the optimality ratio, feasibility ratio, and the normalized risk factor. Depending upon these measures and the concept of possible scenarios, a novel and effective analysis of the problem is done. Unlike other algorithms, the proposed algorithm involves an important role for the decision-maker (DM) in defining a satisfied optimal solution by using a utility function and other required parameters. Numerical examples are used for comparing and illustrating the robustness of the proposed algorithm. Finally, applying the algorithm to treat a Solid Waste Management Planning is introduced. Generally, the data on real-life problems commonly have uncertain behavior . UncertaThere are various methodologies for uncertainty modeling and analysis such as probabilistic methods, possibilistic methods, or both methods . ProbabiRommelfanger  and TanaDo not introduce an optimal solutionIntroduce a set of optimal solutions in the form of the intersection of the decision variables that are represented as intervalsHave fallen to determine the exact set of optimal solutions such that the determined set contains infeasible solutions and/or nonoptimal solutionsHave fallen to determine the exact range of the optimum value except for the BWC methodDo not analyze the risk of selecting one optimal solution from the set of optimal solutionsIgnore the role of the DM in the process of treating the problemThe uncertain nature of ILP problems must be reflected in the optimum value and the optimal solution. Therefore, the ILP problem, in general, has a set of optimal solutions and a corresponding set of optimum values. Jafar et al.  applied The previous failures of other methods represent the research gap in this study and the main motivation for trying to propose a novel method to cover this gap or at least minimize it. After studying the problem, an efficient analysis cannot depend on the classical terminologies about optimality and feasibility such as the optimal solution, the optimum value, and the feasible solution. Therefore, novel terms are introduced such as optimality ratio, feasibility ratio, and risk factor. Moreover, since the terms optimal and feasible solutions are not suitable for the ICLP problem, the terms possible optimal, definite optimal, possible feasible, and definite feasible solutions are used.Moreover, a novel algorithm is proposed for treating ICLP problems where the analyzing process is achieved by simulating different scenarios of the uncertain environment. The scenario method is a common strategy for dealing with uncertain parameters based on scenario analysis . ThroughThe next sections are organized as follows: This section provides an introduction to different forms of the ILP model, some essential definitions, and theorems for the optimal solution set to the ILP model.w±=, where w− ≤ w± ≤ w+, w± ∈ Rn. Besides, an interval number wj± is generally denoted by , where wj−, wj+ ∈ R and wj− ≤ wj+. If wj−=wj+, then wj± will be called interval point. Also, wj denotes a number that belongs to an interval number, i.e., wj ∈ =wj±. Moreover, let w1±=, w2±= be two interval numbers and a ∈ R+, then the basic arithmetic operations on interval numbers can be defined as follows =, −a=, w1± ± w2±=, w1± w2±=, w1±/w2±=.An interval number vector is denoted by  follows –43:a. They represent uncertainty about the corresponding optimum value.Also, for a possible optimal solution x· is formulated as follows:w1, w2, w3, and w4 are random weights that are determined by interacting with the DM such thatIf the ICLP has only one possible optimal solution, then that possible optimal solution is the optimal solution. While in the case of having more than one possible optimal solution, a utility function is proposed to measure the utility of any possible optimal solution. The proposed utility of a possible optimal solution w1 represents the weight of getting an optimal solution that has the largest optimum value,w2 represents the weight of getting an optimal solution that has the smallest difference between the superior and the inferior optimum value,w3 represents the weight of getting an optimal solution that has the largest feasibility ratio,w4 represents the weight of getting an optimal solution that has the largest optimality ratio.Moreover, the attitude of the DM can be represented through the weights, whereBesides, for a decision in an uncertain environment, a risk factor represents the risk of selecting a possible optimal that has a probability of not being optimal or not being feasible. The risk factor can be defined as follows:The risk factor of a definite optimal solution equals zero. Also, practically, a normalized risk factor can be defined as follows:xalt. The set of all alternative optimal solutions is denoted by Θ. Therefore, the satisfied optimal solution is the optimal solution to the following problem:xalt ∈ Θ, w1+w2+w3+w4=1, wi ≥ 0, ∀i.According to the utilities of possible optimal solutions, at least one possible optimal solution can be selected to be the satisfied optimal solution. A possible optimal solution that is a candidate to be the satisfied optimal solution will be called an alternative optimal solution and will be denoted by Z±=,The range of the optimum value is denoted by xj is denoted by xj±=.The solution space is the range of the components of the possible optimal solutions in the form of interval number that the range of components Classical terminology for analyzing the result of solving ICLP is as follows:If exists, the definite-optimal solutionAt least one possible optimal solution with the largest optimality ratioAt least one possible optimal solution with the smallest optimality ratioAt least one possible optimal solution with the largest optimum valueAt least one possible optimal solution with the smallest optimum valueThe superior and the inferior optimum value of at least one possible optimal solutionAt least one possible optimal solution with the largest feasibility ratioAt least one possible optimal solution with the smallest feasibility ratioAccording to interaction with DM, the utility and the risk factor of the alternativesAccording to interaction with DM, the satisfied optimal solution can be determinedAccording to the numerical examples, the notions for determining the solution space are not suitable where the solution spaces are not always convex, connected, or have a fixed shape. Therefore, it is not recommended by the proposed algorithm to use the used notions for the solution space. It will be used in case of a comparison with other algorithms. Instead of determining the solution space, the exact optimal solutions are determined as the set of possible optimal solutions. Besides, novel terminologies can be raised for analysis by using the properties of the possible optimal solutions as follows:All the obtained possible optimal solutions represent possible scenarios which means they can be feasible and optimal. Other techniques depend upon solving transformed models which lead to infeasible solutions such as SOM-2, TSM, and BWC methods, whereas other techniques produce nonoptimal solutions such as ThSM .It treats some or all the obtained possible optimal solutions as the alternatives to the MCDM problem. Therefore, the DM represents the main role in determining the satisfied optimal. By interacting with the DM and according to his attitude, the alternatives and the weights of the utility must be determined.The algorithm ends by determining a satisfied optimal depending upon new analysis with new terminologies while the other ends by determining an expected range of the optimal depending on the classical analysis.Since there is no fixed method to check all possible scenarios, the proposed algorithm offers a better analysis of the model by analyzing the results of a suitable set of possible scenarios. The novelties of the proposed algorithm can be summarized as follows:The suggested algorithm can be described as follows:Reformulate the ICLP model in the form of its equivalent SICLPK possible scenarios where the first three scenarios represent the best, the worst, and the midpoint possible scenarios, respectively.Generate randomly a set of K possible scenariosSolve If the definite-optimal solution exists then it is the optimal solution and then stops. Else, analyzing the set of the possible optimal solutions by determining the superior and the inferior optimum value and the optimality ratioK possible scenarios) and the parameter TInteracting with the DM to determine a set of the alternatives  of the utility function, which satisfy w1+w2+w3+w4=1,  wi ≥ 0, ∀iInteracting with the DM to determine the weights  and how much it consumes the resources. These characteristics represent the main role when interacting with the DM to select a set of them as the alternatives.It must be noted that the attitude of the DM can be represented by the weights. For instance, if the DM is very concerning about the risk factor only, he/she may assign the weights as follows:On the other hand, if he/she is concerned about how large the optimum value is and not concerned about the risk factor, the weights may be assigned as follows:According to the algorithm, the utility is used to rank the alternatives and it is the only measure for determining the satisfied optimal solution.Moreover, For implementing and illustrating the efficiency of the proposed algorithm, a code in Visual Basic is created and implemented on the computer with Intel(R) Core (TM) i3-2330 CPU, 2.2 GHz, and 6 GB RAM. For computational studies, it is supposed to fix the parameters of the proposed algorithm as follows:Three numerical examples are used. The first one represents an ICLP model that has interval coefficients in the objective only while the objective function and the constraints in the second have interval coefficients. The first two problems are used to declare the robust analysis of the proposed algorithm. The third numerical example is assigned for comparing the proposed algorithm and other algorithms. The problem has complete interval coefficients in both the objective and constraints.The following example has interval coefficient at the objective only and is defined as follows:By applying the proposed algorithm, the possible optimal solutions of that example are , , and . Moreover, suppose that all the possible optimal solutions are selected as alternatives. The following example has interval coefficients at the objective and the constraints. It is defined as follows:By applying the proposed algorithm, 64 possible optimal solutions are determined for the following example. Moreover, suppose that five possible optimal solutions are selected as the alternatives as illustrated in Through this example, a comparison between the proposed algorithm and other algorithms is introduced by solving the following problem which is defined as follows:subject toBy applying the proposed algorithm, 100 possible optimal solutions are determined for the following example. Suppose that five alternatives are selected as illustrated in Just for illustration, if the DM is concerned about the risk factor of selecting an alternative and looking for minimizing the risk, he/she can assign the weights as follows:Z±). According to the classical notions of the solution space (x± and y±), all the algorithms failed to determine the exact region of the possible optimal solutions. The proposed algorithm does not depend upon the classical notions of solution space for analysis. It depends upon the generated possible optimal solutions that always belong to the exact region of the possible optimal solutions. The analysis by other algorithms does not lead to the optimal solution for the problem.Moreover, According to the result of solving this example in comparison with other methods, 6t and  t/d, respectively. The WTE facility produces residues of approximately 30% (on a mass basis) of the incoming waste flow. The benefit of WTE is approximate  $/t combusted [In the field of the municipal solid waste (MSW) management system, waste flows delivered to disposal facilities should not exceed their maximum capacities. ombusted , 49.xijk and represent the amount of the waste flows from city j to waste disposal facility i in period k. The application objective is to find minimum system costs through effectively allocating the waste flows from the three cities to the two waste disposal facilities, and the constraints involve the relationships between the decision variables and the waste generation/treatment conditions. The ILP model can be formulated as follows [k± denotes transportation costs of waste flow (from the WTE facility to the landfill in period k) ($/t); OPik± denotes facility operating costs i in period k ($/t); REk± denotes revenue from the WTE facility in period k ($/t); TE± denotes the maximum capacity of the WTE facility (t/d); TL± denotes the capacity of the landfill (t); TRijk± denotes transportation costs from city j to facility i during period k ($/t); WGjk± denotes waste generation rate in city j to facility i during period k (t/d); xijk denotes waste flow rate from city j to facility i in period k (t/d), i = 1, 2; j = 1, 2, 3; k = 1, 2, 3; i denotes index for the facility ; j denotes index for the three cities ; and k denotes index for the time period .The problem under study is to minimize the total system cost and optimally determine the waste flows under many waste disposal constraints and environmental constraints. An ILP model can thus be formulated where the decision variables are denoted as  follows , 49, 50:j to a disposal facility i in a period k must be nonnegative.The overall system cost includes two parts. One part is the transportation cost of waste delivered to the landfill and WTE facility. The second part is the operation costs of the landfill and WTE facility. As for the WTE facility, its revenue should be subtracted as shown in formula . ConstraBy applying the proposed algorithm, the objective is reformulated as follows:Moreover, this application is an illustration of applying the proposed algorithm to real-life problems. The problem has infinite scenarios that need infinite time to be solved. Therefore, the role of DM has main importance for selecting a suitable and satisfactory number of scenarios to be solved. Just for illustration, supposing that after interacting with the analyst, he/she decides to solve 100 possible scenarios that include the best, the worst, and the midpoint scenarios, and the other 97 possible scenarios are selected randomly. Therefore, the parameter K is set to be equal to 100.Going on completing the procedures of the algorithm by solving 100 possible scenarios, 100 possible optimal solutions are determined. The next step is to interact with the DM for illustrating the obtained possible optimal solutions and their properties such as the corresponding optimum values range of the problem (maximum and minimum), the corresponding objective value range of each solution (superior and inferior), the resource-consuming for each constraint for each solution, and the type of the corresponding scenario of each solution. Supposing that, just for simplicity, five possible optimal solutions are selected as the alternatives as illustrated in T. It must be noted that a larger value of T leads to a more confident feasibility ratio, but more time is needed. After calculating the feasibility ratio for each alternative, the DM and the analyst must interact to decide on satisfied weights. Supposing that the assigned weights areAlso, supposing that the DM assigned 1000 for the parameter It must be noted that different assignments for the weights will lead to different satisfied optimal solutions. Therefore, the assigned values are just for illustration. The next step is to calculate the utility for each alternative and comparing among the different alternatives concerning the utility value. Moreover, It does not consider all possible scenarios since they are infinite.Since the vision and interest differ from one DM to another, it is impossible to determine unified a technique for determining the selected scenarios. Therefore, the proposed does not involve a quantitative method for determining the selected scenarios and what is the satisfied number of them.The proposed does not use a quantitative technique for determining the weights. It just depends upon the interaction. But the interaction itself may contain a quantitative technique. It was preferred to concentrate on the main steps for clarifying. The advantages and disadvantages of the proposed algorithm can be stated in The core advantage of the proposed algorithm is that it guarantees to find a satisfied optimal solution, unlike other methods. Also, novel terminology and analysis are used. Moreover, it represents the DM vision of the optimal. But there are some critical limitations of the proposed algorithm.A novel algorithm was proposed to solve the ICLP model depending on simulating a set of possible scenarios. According to the authors' information, the proposed algorithm is the only one that involves the DM in the process of determining the optimal solution which is called the satisfied optimal solution because it concerns the DM vision. Novel characteristics for the solutions are defined by novel terminologies such as the optimality ratio, feasibility ratio, and normalized risk factor. Through interaction with the DM, a set of optimal alternatives is determined from the set of possible optimal solutions. Also, the DM determines his/her weights for a suggested utility function that is used to determine the satisfied optimal solution of the model. Moreover, since the optimal solution of the ICLP problem represents a decision under uncertainty, a suggested normalized risk factor is calculated for measuring the risk of the decision of selecting one possible optimal solution to be the optimal solution of the model. Also, the efficiency and the simplicity of the proposed algorithm are illustrated by numerical examples. The third numerical example with all the coefficients as interval numbers is used for comparing the proposed algorithm and other algorithms from the literature. Finally, the application, Solid Waste Management Planning, is used for applying the algorithm and illustrating its efficiency and reliability. Moreover, through the comparison, the robustness and analysis of the suggested algorithm versus other algorithms are clarified.


There are limited national population-based studies on HPV genotypes distribution in Tunisia, thus making difficult an assessment of the burden of vaccine-preventable cervical cancer. In this context, we conducted a national survey to determine the HPV prevalence and genotypes distribution and the risk factors for HPV infections in Tunisian women. This is a cross-sectional study performed between December 2012 and December 2014. A liquid-based Pap smear sample was obtained from all women and samples’ DNAs were extracted. Only women with betaglobin-positive PCR were further analysed for HPV detection and typing by a nested-PCR of the L1 region followed by next-generation sequencing. A multiple logistic regression model was used for the analysis of associations between the variables. A total of 1517 women were enrolled in this study, and 1229 out of the 1517 cervical samples were positive for the betaglobin control PCR and tested for HPV. Overall HPV infection prevalence was measured to be 7.8% (96/1229), with significant differences between the grand regions, ranging from 2% in the North to 13.1% in Grand Tunis. High-risk HPV genotypes accounted for 5% of the infections. The most prevalent genotypes were HPV 31 (1%), 16 (0.9%), 59 (0.7%). HPV18 was detected only in four cases of the study population. Potential risk factors were living in Grand Tunis region (OR: 7.94 [2.74–22.99]), married status (OR: 2.74 [1.23–6.13]), smoking habit (OR: 2.73 [1.35–5.51]), occupation (OR: 1.81 [1.09–3.01]) and women with multiple sexual partners (OR: 1.91 [1.07–3.39]). These findings underscore the need to evaluate the cost effectiveness of HPV vaccine implementation, contribute to the evidence on the burden of HPV infections, the critical role of sexual behaviour and socioeconomic status, and call for increased support to the preventive program of cervical cancer in Tunisia. Cervical cancer (CC) is the fourth most common cancer among women with an estimated 570,000 new cases in 2018 and the death cause of an estimated 311,000 women worldwide each year—90% of whom live in low- and middle-income countries ), HPV16 (0.9% (11/1229); 95%CI [0.50–1.59]), HPV59 (0.7% (8/1229); 95% CI [0.33–1.27%]) and HPV52 ; 95% CI ) were the most prevalent LR-HPV. The prevalence of the detected HPV genotypes is illustrated on HPV infections were more prevalent in smears with intraepithelial lesions (11.8%), but no significant association was observed between cytological status and HPV infection .HR-HPV genotypes were mostly observed in smears presenting normal cytology (28/49) or having benign transformation (15/37) .HPV16 (14%) and HPV18 (4%) were mainly detected in smears with normal cytology . In a deThe mean age of HPV-positive women was 38.39 ± 1.019 years. HPV prevalence did not vary significantly among the different age groups. However, two prevalence peaks were found: the first in less than 30 years old group with a prevalence of 12.4% ; the second for women over 50 (8.1% (13/160), 95%CI [6.8–19.6%]) .p = 0.08) (Multiple infections were mainly found in young women (under 30) (24%) and women aged between 40 and 50 years old (37%) over 40 years of age. However, this observation was not statistically significant ( = 0.08) .p = 0.002) but the difference was not significant for LR-HPV (p = 0.45).HR-HPV infections increased significantly with age and were more prevalent among women with age bellow 30 years old or over 50 years old . We measp < 10−3).The prevalence of HPV infection by region is shown in the figure below. The highest prevalence was observed in the regions of Grand Tunis  and South-East  . This diWhen we regrouped the different regions to grand-regions; South-East and South-West to South, North-East and North-West to North and Central-East and Central-West to Central, the results remained the same. The lowest value was observed in the grand-region of the North  (95% CI [2.8–14.6%]). The regions of South and Central presented a prevalence of 11.6% (95% CI [7.6–17.2]) and 6.3% (95% CI [4.4–9]), respectively.For the univariate and multivariate analysis, we considered the grand regions: South-East and South-West to South, North-East and North-West to North and Central-East and Central-West to Central.In a univariate analysis, the socio-demographic factors associated with HPV infection were marital status, professional activity, and geographical area (grand-region). Medical history factors significantly associated to HPV infection were history of sexually transmissible infections and parity. Significantly associated behavioral factors were smoking and multiple partners of the women.p-values did not exceed 0.2  and South (13%) while the lowest was observed in the North (2%). This difference was statistically significant. The disparity in HPV distribution could be linked to sexual behaviours and differences in levels of conservative behaviour in the Grand Tunis and North. This latter region could be considered as mostly rural, and it is known that rural communities are typically more conservative than urban communities. Different studies from diverse geographical areas/countries reported that HPV infection prevalence was higher in urban than rural areas ,19,20,21According to previous reviews and meta-analysis, the adjusted global prevalence of HPV infection was estimated at 11.7% and the country-adjusted HPV prevalence ranged from 1.6% to 41.9% worldwide. It was reported that the highest prevalence of HPV infection was observed in some parts of sub-Saharan Africa 24%). Latin America and the Caribbean (16.1%) ,23, in E%. Latin Regarding the age of infected women, we found two peaks: women under 30 (20.0% [8.4–36.9%]) and over 50 years (17.0% [6.8–19.6%]. The second peak observed in our study has been described in most developed countries ,30,31,32The HPV genotypes distribution obtained in this study agrees with the widely reported data, clearly showing that HPV16, 31, 59 and 52, the most oncogenic HPVs, are the prevalent genotypes in Tunisia. This finding may encourage the introduction of the Nona-valent HPV prophylactic vaccine that preferentially targets these HR-HPVs genotypes . One impFrom a cytological point of view, HPV was identified in only 11.8% of samples with precancerous lesions. This can be due to the low specificity of Pap smears reading or to a low viral charge, knowing that the viral charge decreases with the lesion progression ,37. On tAs regards to risk factors, the multivariate model used in our study population retained geographical region, occupation, marital status, smoking and sexual habits of women as potential risk factors by adjusting for other potential confounders. According to the literature, the main risk factors associated with HPV infection in women are age, socio-demographic characteristics, sexual behaviour, history of sexually transmitted infections, parity, contraceptive methods, and smoking ,41,42. MThis study reports a 7.8% HPV prevalence in Tunisian women with significant differences between regions. A high rate of HR-HPV infection has been identified. HPV16 and 31 were the most prevalent, whereas HPV 18 was detected in only four cases. It is important to take into consideration that HPV18 was one of the most frequent in invasive cervical carcinoma and despite its little prevalence in this study remains one of the most oncogenic genotypes. These findings are important for the update and the optimization of preventative strategies against HPV infections and cervical cancer in Tunisia. The introduction of the Nona-valent vaccine could strengthen the prevention of cervical cancer in Tunisia. Its implementation should be discussed considering its cost-effectiveness and the targeted population in relation to circulating HPV genotypes in Tunisia.


This cohort study examines the association between the Advisory Committee on Immunization Practices recommendation update for patient-clinician shared decision-making and human papillomavirus vaccine administration among US adults aged 27 to 45 years. What are the trends of human papillomavirus (HPV) vaccine administration among US adults aged 27 to 45 years with commercial insurance before and after the Advisory Committee on Immunization Practices recommendation update for patient-clinician shared decision-making?In this large commercial claim-based cohort study of 22.6 million enrollees aged 27 to 45 years in the US, the HPV vaccine administration rates in both women and men showed a statistically significant increase associated with the age-expanded Advisory Committee on Immunization Practices recommendation update.Further research is warranted to explore the decision-making process in receiving HPV vaccination to maximize the cancer prevention benefit in this age group. In 2019, the Advisory Committee on Immunization Practices (ACIP) recommended patient-clinician shared decision-making for human papillomavirus (HPV) vaccination in adults aged 27 to 45 years. Less is known about the HPV vaccine administration trends in this age group before and after this recommendation update.To examine the association between the ACIP recommendation update and the HPV vaccine administration among US adults aged 27 to 45 years.This large commercial claim-based retrospective cohort study used the Optum Clinformatics database for validated claims from January 1, 2007, through December 31, 2020. A total of 22 600 520 US adults aged 27 to 45 years without previous HPV vaccination claims during the study and enrollment period were included.The first-appearing HPV vaccination claim per individual was defined as a new HPV vaccine administration. Interrupted time-series analyses were conducted to assess the association between the ACIP update and the quarterly vaccine administration rate change. The annual rate trends across race and ethnicity groups and the proportions of vaccination cases by sub–age groups and valent types were also estimated. Vaccine administration trends were assessed by race and ethnicity in this age group because HPV vaccination trends were found to differ by race and ethnicity in the initially eligible population.β2, 40.18 per 100 000 persons; P = .01) and an increased slope  over time postupdate. The ACIP update was only associated with an immediate increase in vaccine administration in men . The annual rate trends were similar across race and ethnicity groups. Age at vaccine administration shifted over time . The most administered HPV vaccines in 2020 were 9 valent .Among 22 600 520 final study participants, the majority were men (50.9%) and non-Hispanic White (53.4%), and the mean (SD) age when first observed was 34.6 (5.8) years. In women, the ACIP update was associated with an immediate increase in vaccine administration rate (coefficient In this population-based cohort study, there were statistically significant increases in HPV vaccine administration in adults aged 27 to 45 years after the ACIP recommendation update. Patient-clinician shared decision-making may have been the main associated factor for this increase. Further research is warranted to explore the decision-making process in receiving HPV vaccination and to develop effective decision aids to maximize the cancer prevention benefit in this age group. Additionally, recent studies report that oropharyngeal cancer and anal cancer incidence rates are increasing by nearly 3% annually in the US.3Human papillomavirus (HPV) causes cervical, oropharyngeal, anal, penile, vaginal, and vulvar cancers and is associated with approximately 45 000 new cancer diagnoses annually in the US.7 Therefore, the US Advisory Committee on Immunization Practices (ACIP) recommends HPV vaccination for those aged 9 to 26 years (since 2006 for women and 2011 for men).9 The ACIP guideline currently recommends routine vaccination for those ages 11 to 12 years and catch-up vaccination through age 26 years.7 In October 2018, the US Food and Drug Administration (FDA) expanded the approved age range for the use of the 9-valent HPV vaccine to ages 27 to 45 years in both women and men.10 The ACIP recommended patient-clinician shared decision-making for HPV vaccination in this newly approved age group in June 2019.7The HPV vaccine is an effective prevention against HPV-associated cancers, especially when administered before exposure to HPV types covered in the HPV vaccine.12 evidence supports vaccine efficacy in previously unexposed older populations.14 Therefore, some of these individuals may feel the need to get vaccinated for protection against HPV. Patient-clinician shared decision-making based on the guideline update may also have changed HPV vaccine administration in this age group, as evidence shows shared decision-making improves vaccination in general.16 Moreover, most commercial insurances cover vaccination for those recommended by the ACIP17; thus, there may be an expected trend change in the HPV vaccination rate in this age group after the FDA approval and the ACIP recommendation update.Individual differences in previous exposure to HPV and clinical characteristics affect the effectiveness of the HPV vaccine in this older age group. In fact, though it is reported that HPV vaccination beyond 26 years old is not cost-effective at the population level at current prices,19 We additionally assessed how the makeup of ages when administered and valent types of the vaccine shifted over the years.However, there is a lack of evidence-based assessment of such trend change. Therefore, this cohort study aimed to determine the temporal trend of the HPV vaccine administration rate and the association between the ACIP recommendation update and the vaccine administration change among adults 27 to 45 years old who were enrolled in commercial insurance from 2007 to 2020. We also assessed if vaccine administration trends differed by race and ethnicity in this age group because HPV vaccination trends were found to differ by race and ethnicity in the initially eligible population.20 Demographic information—including age, sex, and race and ethnicity—of the plan members, dates of medical service, diagnosis  and procedures , and payment information are available.For these analyses, we conducted a retrospective cohort study using the Optum Clinformatics database for validated claims from January 1, 2007, through December 31, 2020. The Clinformatics data set covers approximately 64.3 million lives enrolled in commercial health plans and comprises deidentified medical and pharmacy claims from all 50 US states. Individuals are given unique identifiers that allow researchers to follow individuals as they enroll, disenroll, and re-enroll in the health plan.STROBE) reporting guideline for cohort studies.This study was deemed exempt from review by the institutional review board of The University of Texas Health Science Center owing to use of deidentified data. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology .22 This algorithm has demonstrated 97% specificity, 48% sensitivity, and 71% positive predictive value for estimating the race of Black individuals.23 The sex variable is categorized as binary .In this study, we investigated the vaccine administration trend, defined as the rate of having at least 1 HPV vaccination claim (the first-appearing HPV vaccination claim) during the study period among the eligible population. The study aimed to identify the general trends in the HPV vaccine administration itself among newly vaccine-approved older age groups rather than their up-to-date vaccine coverage. The total study population (the denominator) was identified as adults aged 27 to 45 years for each enrollment time period (either year or quarter) who did not have any HPV vaccination claims in the past time periods of their enrollment. For example, if an individual had the first-appearing HPV vaccination claims in 2010, we excluded the individual from the denominator in calculating the HPV vaccination rate in 2011 and thereafter. We identified the administration of the HPV vaccine by 24 To assess the shifts in the distribution of age at administration in these older adults, we calculated the proportions of sub–age groups by year  among the vaccinated participants. We also calculated the proportions of valent types of HPV vaccine administered per year.Descriptive statistics were used to summarize the characteristics of the final study population of adults aged 27 to 45 years. To quantify exploratory trends in annual administration rates and determine any trend change in the study period  by sex and race and ethnicity, we used the joinpoint regressions.25 This analysis evaluates the immediate level change (difference in rates) before and after the guideline update and the postupdate trend change over time (difference in slopes). Data were grouped quarterly to test the hypothesis. We hypothesized a trend break in the second quarter of 2019 (ACIP update) for the base-case analysis. Additionally, we tested a trend break in the fourth quarter of 2018  to assess if FDA approval was associated with any change. Detailed methods are explained in eMethods 1 and 2 in the Finally, we conducted an interrupted time-series analysis to assess the effect of the ACIP recommendation update.To address several limitations of the nature of claims data, we performed 3 different sensitivity analyses using annual information. In the Clinformatics database, the enrollee’s age was calculated by subtracting birth year from the claim year, which could result in a 1-year difference . Some of the 27-year-olds with vaccine claims might also have initiated the series of vaccination when they were 26 years old while enrolled in another plan. Therefore, we performed a sensitivity analysis  that excluded those aged 27 years from the study sample to address these concerns. We conducted sensitivity analysis 2, where we only included the birth cohorts that were never eligible for the HPV vaccine until 2018’s new FDA approval (women born in or before 1979 and men born in or before 1984 among the ages up to 45 years every year). Because these birth cohorts were not eligible for the HPV vaccine until the new approval, they had a minimal likelihood of getting vaccinated while enrolled in another plan. For the primary analysis, we did not restrict the study samples based on the enrollment period because the main interest in this study is new HPV administration rather than up-to-date vaccination. To test the robustness of the main findings, we conducted sensitivity analysis 3, only including those fully enrolled for each calendar year.P < .05, and all hypotheses were 2-sided. We used Joinpoint Trend Analysis software, version 4.9.0.1 ; SAS, version 9.4 (SAS Institute); and Stata, version 17.0 (StataCorp) for all analyses.All analyses were conducted stratified by sex. Statistical significance was assessed at Among 22 600 520 final study participants (65 043 302 person-years), 50.9% were men and 53.4% were non-Hispanic White; 18.6% of study participants had missing information on race and ethnicity. The mean (SD) age when first observed was 34.6 (5.8) years . BetweenP < .001), then became stagnant until 2018 . After 2018, there was a statistically significant increase in the administration rate . The administration rates were consistently low for men, with no change from 2007 until 2009 . From 2010, it increased by 3.0 per 100 000 persons annually until 2018; after 2018, there was a statistically significant increase in the rate, as in women . There were similar patterns across the race and ethnicity groups in both men and women  and an increased slope over time postupdate . The ACIP update was only associated with an immediate increase in vaccine administration in men . When using the FDA approval as a trend break, it was associated with a slope increase in women  but both an immediate level increase  and slope increase  in men.Results from interrupted time-series analyses using quarterly data showed that the ACIP update was associated with improved HPV vaccine administration rates . In womeP < .001) and men . Sensitivity analysis 2, which only included the birth cohorts that were never vaccine eligible until the new approval, showed similar statistically significant increasing trends after 2018 in both women  and men  (P < .001) and men , supporting the robustness in the present findings  . Sensitifindings .In this population-based cohort study, we found that there was a statistically significant increase in the HPV vaccine administration rate after 2018 among adults aged 27 to 45 years. Women and men showed distinct patterns before 2018, majorly due to the different timings of HPV vaccine recommendations. In this hypothesis-driven analysis, the results showed that the ACIP recommendation change was statistically significantly associated with the increase in HPV vaccine administration rate in this population.19 However, this study did not find statistically significant differences in administration trends among newly recommended ages. Evidence suggests racial and ethnic disparities in HPV-associated cancer incidence.27 Further research is needed on how catch-up vaccination in this age group can help reduce these existing disparities.The HPV vaccination rate among those aged 9 to 26 years showed considerable disparities across racial and ethnic groups.In 2019 and 2020, those aged 31 to 45 years made up the majority of the vaccinated adults aged 27 to 45 years. This makeup was the opposite until 2018, whereas the majority of the cases were among the age group of 27- to 30-year-olds. These individuals might have missed their vaccination schedule when they were still eligible and now have received catch-up vaccination as they became eligible again. On the other hand, many of these individuals aged up to 45 years who received the vaccine were never eligible for the initial ACIP HPV-vaccine recommendation . Shared decision-making may have been effective for those who were never eligible but had perceived benefits of the HPV vaccine.28 and as of late 2016, only the 9-valent vaccine has been distributed in the US.7 Because some adults without adequate vaccination might be at risk for a new HPV infection targeted by the 9-valent vaccine, HPV vaccination may benefit these individuals.The dose-specific proportions can be explained by the availability of the vaccine and the ACIP recommendation. In the US, all 2-, 4-, and 9-valent types are approved for use among women, while only 4 valent and 9 valent are approved for use in men. The 4-valent HPV vaccine has been available and recommended since 2006,30 The likelihood of receiving the HPV vaccine was highly associated with the perceived likelihood of benefiting from the vaccine.29 Similarly, according to a study that surveyed primary care physicians, the majority of physicians reported that they would be more likely to recommend HPV vaccination to adults aged 27 to 45 years due to the new ACIP recommendation.31 Evidence also supports the substantial effectiveness of patient-clinician shared decision-making in vaccination, cancer prevention, and other health outcomes in general.33 Especially because the HPV vaccine has been available for more than a decade, shared decision-making may benefit those interested in vaccination based on their perceived benefit but who missed their vaccination schedule or were not eligible. However, also the majority of the physician respondents were unsure of what to emphasize during the shared clinical decision-making regarding HPV vaccination.31 The present findings showed a statistically significant increase in HPV vaccination in this age group after the ACIP update, yet they make up very low rates. These studies together represent the great interest in HPV vaccination among both adults aged 27 to 45 years and health care professionals, as well as the potential effectiveness of their shared decision-making, though there remains a need for further research on enhancing this process .34Previous studies that surveyed adults aged 27 to 45 years after the ACIP recommendation update found that the majority of these adults were likely to ask health care professionals about HPV vaccination and willing to get a vaccine if recommended by their clinician.35 Effective shared decision-making for selective age-expanded HPV vaccination most likely involves a conversation about the patient’s sexual history. However, according to a study in 2020, 65% of primary care visits did not involve sexual history taking.36 The sexual history taking based on the validated model, despite being recommended by the Centers for Disease Control and Prevention, only comprised 1% of the visits.36 In 2020, the US Preventive Services Task Force updated its guideline on behavioral counseling interventions for reducing sexually transmitted infections.37 To successfully implement this guideline, it is crucial for health care professionals to initiate a conversation about the patient’s sexual history.38 In addition, it is necessary that the health care professionals recognize the diversity of gender identities and sexual orientations for more effective sexual history taking39 and shared decision-making in HPV vaccination. Although this US Preventive Services Task Force guideline is not exclusively for HPV vaccination in older adults, it could provide an opportunity to identify those who might benefit from the age-expanded HPV vaccination.The current updated recommendation for age-expanded HPV vaccination provides an opportunity to prevent cancers in those who are at risk of new HPV infection and were not adequately vaccinated. Proper shared decision-making that allows health care professionals to discuss HPV vaccination with individuals who are most likely to benefit is key to maximizing the cancer prevention effects. This process is particularly needed to decide who should be prioritized because the universal vaccination is not cost-effective in this age group and there is a limited vaccine supply.29 Due to the information asymmetry between clinician and patient regarding patients’ sexual history and other related clinical factors, patient-initiated shared decision-making might be meaningful in age-expanded HPV vaccination. However, previous nationally representative studies found that US adults aged 27 to 45 years had a low level of awareness and knowledge about HPV-related information.41 For patient-initiated conversation about HPV vaccination, disseminating information about the recommendation update and vaccine benefits is instrumental in reaching more people who likely will benefit from vaccination.In recognizing the challenges from the health care professionals’ side to identify the population that would most likely benefit from the age-expanded catch-up vaccination, it is equally important that patients initiate the shared decision-making. The literature indicates the patients’ awareness of age expansion, belief in vaccine effectiveness, and perceived benefit of HPV vaccination were associated with the likelihood of asking their clinicians about the HPV vaccination in this age group.42 and it warrants further research in HPV vaccine utilization across race and ethnicity among 27- to 45-year-olds. We also acknowledge the limitation of the claims data only with a binary sex variable. Gender identity, along with sexual orientation, can be an important factor in HPV vaccination with a possibly different burden of HPV.45Limitations of this study include the sample of commercially insured adults, which limits the generalizability of the findings to all US adults in this age range. Due to the nature of insurance claims data, this study is also subject to limitations such as loss to follow-up (lack of utilization information before or after the enrollment) and incomplete or inaccurate administrative coding. Derivation of race and ethnicity information is also subject to limitations due to the limited validity of its method,49 and the longitudinal information with dates of health care utilization allowed us to identify the exact timing of HPV vaccine administration and avoid biases related to self-report.Despite these limitations, this study adds to the literature by providing an analysis of large numbers of adults in the US. Claims data are utilized often in vaccination administration studies,This cohort study found statistically significant increases in HPV vaccine administration in adults aged 27 to 45 years after the FDA approval of use and the ACIP recommendation update. For maximizing the cancer prevention benefit in this age group, effective patient-clinician shared decision-making is crucial. Future studies are needed to address the difficulties surrounding identifying those who will most likely benefit from the vaccine and the unsureness of what to emphasize during shared decision-making. Developing effective decision aids would be the key to successfully implementing this updated ACIP guideline.


The Human Papillomavirus (HPV) test is a crucial technology for cervical cancer prevention because it enables programs to identify women with high-risk HPV infection who are at risk of developing cervical cancer. Current U.S. Preventive Services Task Force recommendations include cervical cancer screening every three years with cervical cytology alone or every five years with either high-risk HPV testing alone or high-risk HPV testing combined with cytology (co-testing). In Argentina, 7,548 new cervical cancer cases are diagnosed each year with 3,932 deaths attributed to this cause. Our study aims to show the clinical implementation of a cervical cancer screening program by concurrent HPV testing and cervical cytology (co-testing); and to evaluate the possible cervical cancer screening scenarios for Latin America, focusing on their performance and average cost. A cervical cancer screening five year program via co-testing algorithm (Hybrid-2-Capture/cytology) was performed on women aged 30–65 years old at a university hospital. Statistical analysis included a multinomial logistic regression, and two cancer screening classification alternatives were tested (cytology-reflex and HPV-reflex). A total of 2,273 women were included, 91.11% of the participants were double-negative, 2.55% double-positive, 5.90% positive-Hybrid-2-Capture-/negative-cytology, and 0.44% negative-Hybrid-2-Capture/positive-cytology. A thorough follow-up was performed in the positive-Hybrid-2-Capture group. Despite our efforts, 21 (10.93%) were lost, mainly because of changes on their health insurance coverage which excluded them from our screening algorithm. Of the 171 women with positive-Hybrid-2-Capture results and follow-up, 68 (39.77%) cleared the virus infection, 64 (37.43%) showed viral persistence, and 39 (22.81%) were adequately treated after detection via colposcopy/biopsy of histological HSIL . The prevalence of high-risk HPV in this population was 192 women (8.45%), with HSIL histology detection rates of 17 per 1,000 screened women. A multinomial logistic regression analysis was performed over the women with positive-Hybrid-2-Capture considering the follow up  as dependent variable, and the cytology test results  as independent variable. The model supported a direct association between cytology test results and follow up: negative-cytology/clearance, ASC-US/persistence, and positive-cytology/HSIL with the following probabilities of occurrence for these pairs 0.5, 0.647 and 0.647, respectively. Cytology could be considered a prognostic-factor in women with a positive-Hybrid-2-Capture. These findings suggest that the introduction of co-testing could diminish the burden of cervical cancer in low-and middle-income-countries, acting as a tool against inequity in healthcare. In 2020, cervical cancer was the fourth most common cancer in women globally, with an incidence of 13%, resulting in 341,831 deaths worldwide, over 90% of these occurring in low- and middle-income countries (LMIC). Almost 86% of cases and 88% of deaths occur in developing countries, resulting in the third cause of death among women in Latin America [The International Agency for Research on Cancer (IARC) has classified 12 Human Papillomavirus (HPV) types as group 1 carcinogens considering them pathogenic or high-risk HPV due to their link to the development of cervical cancer –5. AlthoAlthough it can be almost entirely prevented, cervical cancer remains a significant public health problem in Argentina, with 7,548 new cases diagnosed yearly and 3,932 deaths attributed to this cause . In addiNon-pregnant women between the ages of 30 and 65 with no prior history of high-grade cervical lesions (reported in the last 20 years of their lives) and no clinical findings of any HPV related condition at the time of screening, who usually seek medical attention at the hospital, were enrolled prospectively between July 2013 and June 2018 at a University Hospital . This prThe conventional Papanicolaou smear samples were stained and examined under an optical microscope. It is a multichromatic stain that uses five dyes to stain various components of the cells differentially: hematoxylin; Orange G; Eosin Y; Light Green SF yellowish; and Bismarck brown Y, in 95% ethyl alcohol. The stain should result in fairly transparent cells so we can interpret even thicker specimens with overlapping cells . ColposcThe cervical samples for viral determination (Digene HPV HC2 DNA Test®) were taken with Digene Cervical Sampler®, placed in a transport medium, preserved adequately, and processed using the Hybrid-2-Capture technique . The cutoff value in the Relative Light Unit/cutoff ratio (RLU/CO) was ≥1 pg/mL for 13 high-risk HPV types. Hybrid-2-Capture technology detected RNA:DNA hybrids using signal-amplified, chemiluminescent technology, with full genome probes complementary to HPV DNA. The resulting signal was automatically interpreted using the DML instrument with the Digene Hybrid Capture System Version 2 Software. According to the co-test results and following the screening algorithm , women w2χ test. Companion alluvial plot for data distribution was also created, to get a graphical cohort distribution picture. Multinomial logistic regression was used to assess the cytology impact over the follow-up outcome for clearance or histologic HSIL (HSIL-h) compared to the persistence counterpart. Finally, classification performance statistics were calculated for the proposed screening strategies: HPV-cytology (co-testing), serialized HPV-cytology, and serialized cytology-HPV testing. The performance measurements included: sensitivity, specificity, positive/negative predictive value, positive/negative likelihood ratio and diagnostic odds ratio. The diagnostic odds ratio will be the key metric to test the effectiveness of screening strategies proposed in this manuscript as diagnostic tests. It is calculated as the positive likelihood ratio divided by the negative likelihood ratio. As such it is an odds ratio, where the greater the number the better the diagnostic test, from a medical perspective. All statistical tests, except where noted, were two-sided with a statistical significance α = 0.05 and were performed using R version 4.0.5. Complete cohort database can be found in Cohort descriptive statistical analysis over the age and turnaround time included variable range, mean and standard deviation over decade groups . Analysis of variance (ANOVA) test was performed over turnaround time group means. Posterior Fisher’s Least Significant Difference test was performed with Bonferroni p-value correction for multiple comparisons. Homogeneity of variances was tested with Bartlett’s test. Co-test results were presented as contingency tables over the decade groups, and analyzed with A total of 2,273 patients  met the eligibility criteria and were included in the study, with a mean age ± SD of 44.95±9.84 years old, and a mean turnaround time ± SD of 19.31±9.34 days. Independent analysis of the two methods showed that 192 women (8.45%) had positive-Hybrid-2-Capture (HC2); and 2,205 (97.01%), 28 (1.23%) and 40 (1.76%) women had negative, Atypical Squamous Cells of Undetermined Significance (ASC-US), and positive-cytology results respectively. For the data analysis, we decided to separate the ASC-US cytology because of the varying risk associated with this particular cytology according to age and high-risk HPV status . The stuIn order to get a clear picture of the five-year patient follow-up, we constructed an alluvial plot . Each liTo explore the existence of cytology test results with the five-year follow-up we constructed a multinomial logistic regression model with 171 women with positive-Hybrid-2-Capture results . The modIn order to assess the classification performance of the proposed screening strategies for cervical cancer, we had to transform the original results obtained from Of all positive-Hybrid-2-Capture patients, 22.81% had an HSIL histological diagnosis which was timely and adequately treated; 37.43% showed viral persistence, and 39.77% viral clearance; these results differ from other published studies . NeverthWe have determined that the best scenarios included: a) standalone Hybrid-2-Capture  or b2) negative co-tests defined as only double negative cases . EvidencIn Latin America, decades of cytology-based screening to detect precancerous cervical lesions have not made a significant impact in the reduction of cervical cancer incidence, morbidity, and mortality rates , 19, 20.Over the last decade, diverse experiences with HPV testing have been conducted in Latin American countries, some as part of research studies, and others, more recently, to pilot the implementation of HPV testing in the public health system . ArgentiTo obtain the most reliable data, we have excluded patients who reported diagnosis of high-grade lesions in the last 20 years or had clinical findings of any HPV related condition before screening. This has led to significantly less enrollment in comparison to other similar studies. However, the positive-Hybrid-2-Capture rate was 8.45% which correlates with published evidence , 23. ThiOur study was conducted mimicking the National Health Program that used hybrid capture. This technology was chosen for two reasons; on the one hand, for its safety aspects regarding self-sampling. On the other, in 2013, when our study began, hybrid capture was the only clinically validated HPV test available.et al. identified women who died of cervical cancer and multiple women with high-grade lesions who tested negative for L1 but positive for E6/E7. To prevent false negatives caused by gene deletions, the hybrid-capture method uses full genome RNA probes [Currently, the two most used methods for HPV DNA detection are hybridization which uses a whole genome probe, and Polymerase-Chain-Reaction which uses L1 amplification. We chose Hybrid-2-Capture for several reasons; first and foremost, the L1 gene can be deleted in cervical cancers and lead to false-negative results. Tjalma A probes , 25. AlsA probes .The shift from cytology-based screening to molecular HPV testing has required a steep learning curve for screening programs and the laboratories that support them . Latin AFor us, “a woman with cervical cancer is a sign of a fragile healthcare system”. We analyzed the performance of the three possible scenarios for cervical cancer screening in Latin America: cervical cytology (HPV-reflex), HPV testing (cytology-reflex), or HPV testing combined with cytology (co-testing). Their statistical analysis, as well as their performance and cost, determined that any of them could be used under different circumstances for the three patient profiles described for Latin America: i) women with favorable socioeconomic and cultural profiles who are most often diagnosed early; ii) women with a social vulnerability profile whose diagnosis is often too late, and iii) women with profound geographical barriers who will never know their cause of death . In the Even in this era of developmental technology, there are still several countries where women suffer and die from preventable diseases, such as cervical cancer. The five-year cumulative incidence rate of the high-grade lesion of our population was 1.73%, with a 8.45% hrHPV prevalence. The follow-up analysis of the study population at the end of the five years determined that 22.81% of the HC2-positive group had an HSIL histological diagnosis which was adequately treated; 37.43% had viral persistence, 39.77% had viral clearance. Our study provides critical, real-world evidence which could encourage low- and middle-income countries to incorporate co-testing as a cervical cancer screening tool. Multinomial logistic regression analysis supported a direct association with prognosis, i.e., negative-cytologyt/clearance, ASC-US/persistence, and positive-cytology/HSIL. If these findings are confirmed in more extensive population studies, cytology could be regarded as a prognostic factor on Hybrid-2-Capture positive women in a co-testing context. This evidence suggests that the introduction of screening via co-testing in low- and middle-income countries could diminish the burden of cervical cancer, acting as a tool against inequity in healthcare.S1 File(XLSX)Click here for additional data file.