Skin colonization by human papillomavirus (HPV) is typically related to inconspicuous cutaneous infections without major disease or complications in immunocompetent individuals. However, in immunosuppressed patients, especially organ transplanted recipients, cutaneous HPV infections may cause massive, highly spreading and recurrent skin lesions upon synergism with UV-exposure. Current HPV prophylactic vaccines are not effective against cutaneous HPV types (cHPV). By applying a modular polytope-based approach, in this work, we explored different vaccine candidates based on selected, tandemly arranged cHPV-L2 epitopes fused to thioredoxin (Trx) as a scaffold protein. Upon conversion to heptameric nanoparticles with the use of a genetically fused oligomerization domain, our candidate Trx-L2 vaccines induce broadly neutralizing immune responses against 19 cHPV in guinea pigs. Similar findings were obtained in mice, where protection against virus challenge was also achieved via passive transfer of immune sera. Remarkably, immunization with the candidate cHPV vaccines also induced immune responses against several mucosal low- and high-risk HPV types, including HPV16 and 18. Based on cumulative immunogenicity data but also on ease and yield of production, we identified a lead vaccine candidate bearing 12 different cHPV-L2 epitopes that holds great promise as a scalable and GMP production-compatible lead molecule for the prevention of post-transplantation skin lesions caused by cHPV infection. About 5% of all cancers globally result from HPV infection2, with estimates that 604,000 women3 and 60,000 men2 develop HPV-related cancers annually. Over 200 different HPV types are currently described with varying tropism for mucosa or skin4. The skin of all individuals constantly harbors a plethora of HPV, which are rarely associated to malignant pathologies. A number of HPV phylogenetically classified into the beta-genus (beta-HPV) have been assigned as commensals, because of their ubiquitous presence (>90%) in skin swabs or hair bulbs of healthy individuals with no visible lesions or warts8. In fact, the vast majority (>95%) of all viral DNA sequences found in skin samples correspond to papillomaviruses, mostly from the beta- and the gamma-genera9. The controlled adaptation to commensal cutaneous HPV can be, however, de-regulated under immune suppression conditions, as commonly observed in solid organ transplant recipients (OTR)11.The carcinogenic role of some human papillomavirus types (HPV) has been firmly established10. Over 90% of the OTR are reported to acquire cutaneous warts within five years post-transplantation11. Although the mortality associated to these post-transplantation skin lesions is low12, the ablative therapies needed for treatment are painful, disfiguring and may not provide a long-lasting cure given the recurrent characteristics of these skin lesions in OTRs13.The occurrence of cutaneous warts in OTR has been shown to tightly correlate with the duration of iatrogenic immune suppression15. A subgroup of NMSC classified as squamous cell carcinoma (SCC), represents the most common malignancy in this patients’ cohort, with a 60- to 250-fold increased risk of incidence compared to the general population18. In OTR, SCC represents the most aggressive outcome with a considerably higher risk of metastasis and death compared to the general population19. These clinical observations were, in fact, preceded by the earlier discovery of beta-HPV  in SCC from patients affected by a rare genetic skin disease known as epidermodysplasia verruciformis (EV), which is characterized by increased susceptibility to widespread infection by beta-HPV, mainly on sun-exposed sites. A close clinical and morphological resemblance between the SCC observed in OTR and the HPV-induced warts present in EV-patients has also been documented20. Non-classical, acquired forms of EV have been also documented in HIV/AIDS patients21 and in other inherited immune dysfunctions22. Nevertheless, beta-HPV were also detected in NMSC of non-EV patients, with a higher incidence in transplanted patients compared to the general population24. Serological studies substantiated these findings by correlating the development of cutaneous squamous cell carcinoma in OTR to the presence of antibodies against not only beta-26 but also gamma-HPV types 27. Alpha-HPV types known to colonize the skin  have also been associated to cases of widespread, potentially disfiguring cutaneous HPV infections, a condition known as generalized verrucosis commonly observed in patients with underlying immunodeficiency status 29. In our opinion, this scenario illustrates the need to develop a broadly immunogenic vaccine, rather than focusing on a subset of types clustered in a single papillomavirus genus.In addition to a considerable patient morbidity, the profuse and recurrent cutaneous warts observed in OTR are frequently accompanied by keratinocyte-associated malignant lesions known as non-melanoma skin cancer (NMSC)30. A solid organ transplant in the United States may range from USD 442,000 for a single kidney transplant to over 2.6 million for combined heart and lung transplantation31. In Germany, around 3000 people are subjected to chronic immune suppression every year due to organ transplantation32, while the long-term oral maintenance immunosuppression has been estimated to cost USD 2500 monthly per patient33. These expenditures are augmented by the frequent development of skin lesions in OTRs. Policy makers in the United States and United Kingdom recommend that OTRs should be seen in dedicated surveillance dermatology clinics, given the high association of HPV-related warts and NMSC in these patient cohort. About 80% and 54% of transplanted patients in Australia34 and the United Kingdom35 are diagnosed with NMSC within 20 years of immunosuppression following transplantation. In the United States, the annual expenses for NMSC-related medical care are estimated to cost USD 650 million36.According to the global database on donation and transplantation, over 150,000 solid organs were transplanted globally in 2019, which represents a 5% increase compared to the previous year37, which due to its higher sequence conservation can afford a much broader (cross-)protection against multiple HPV types. This paved the way to the development of PANHPVAX38, an L2-based vaccine candidate currently undergoing a first-in-human, single center, open-label, phase I, dose escalation trial in healthy volunteers (NCT05208710). The antigen will be formulated with cyclic-di-AMP as adjuvant. Following the same modular polytope-based approach applied to PANHPVAX, we generated a number of antigens displaying several conserved cHPV L2 epitopes grafted to the display site of a thermally stable thioredoxin (Trx) scaffold protein from the archaeon Pyrococcus furiosus, which was then further assembled into more immunogenic heptameric nanoparticle structures. Herein, we systematically investigated the immunogenicity of a series of candidate polytopic vaccines targeting a panel of cutaneous HPV types clinically associated to post-transplantation cutaneous lesions. We thus identified a leading vaccine candidate bearing 12 different cHPV-L2 epitopes that induce neutralizing antibodies against 19 cHPV in guinea pigs, affords protective immunity in a murine challenge model of HPV infection, and is amenable to a GMP-compatible production.Currently available HPV vaccines based on virus-like particles formed by the major capsid protein L1 are highly safe and effective, yet restricted to a type-specific prevention only acting against low- and high-risk mucosal HPV types associated to anogenital cancer. In recent years, we have documented the immune reactivity and vaccine translational potential of the minor capsid protein L238. Nevertheless, we reasoned that due to the great heterogeneity and phylogenetic diversity of cutaneous HPV the protection afforded by PANHPVAX may not be sufficiently comprehensive  and three  with further expanded polytopes  cutaneous HPV types induced by the PfTrx-L2c12merOVX313 and PfTrx-L2c14merOVX313 candidate vaccines, respectively Fig. . Importap-value = 0.055 as determined by the nonparametric Mann–Whitney test; see Fig. Next, we used two different mouse challenge models to assess vaccine-induced protection via passive transfer of immune sera, focusing on three subsets of cHPV types: (i) HPV types 4, 38, and 76 against which, rather weak antibody titers were previously detected with the L1-PBNA; (ii) HPV types 5 and 21 against which, relatively strong antibody titers were found; (iii) HPV16, representing high-risk mucosal types to which antibody titers were not characterized. In this experimental set-up, passively transferred sera of guinea pigs immunized with the PfTrx-L2c12merOVX313 antigen proved capable of significantly reducing the PSV transduction signal derived from the cervico-vaginal challenge with HPV4 Fig.  and cutaPfTrx-L2c12merOVX313 afforded a lower mean average radiance in animals challenged intra-vaginally with HPV4    Fig. , althougMultiple factors, including ease of production and scalability, influence vaccine developability and potential for translation to a clinical setting. The immunogenicity of PfTrx-L2c12merOVX313 and PfTrx-L2c14merOVX313 antigens were found to be overall quite comparable. In guinea pigs, the latter antigen-induced neutralizing antibodies against 18, rather than 16 cHPV types, but the resulting sera failed to provide protection against cutaneous challenge with HPV4 and a similarly inferior protection compared to PfTrx-L2c12merOVX313 was observed with mice immune-sera. However, an important favorable feature of the 12mer antigen was its higher production yield and ease of purification. In fact, PfTrx-L2c12merOVX313 not only was expressed at higher levels compared to 14mer antigen, but it was also more amenable to a GMP-compatible, heat-treatment and cation-exchange chromatography-based purification, with a final yield of homogenously purified protein  almost 10-fold higher than that achieved with PfTrx-L2c14merOVX313  that are conserved within the L2 proteins of all known papillomaviruses. The display of L2 aa 17–36 region (‘RG1’ epitope) into chimeric HPV VLPs was shown to induce cross-neutralizing responses in rabbit42. Nevertheless, an L2 epitope from a single HPV type does not provide a sufficiently comprehensive coverage44. For this reason, we employed a polytope design for comprehensive coverage of a large panel of different HPV types.The use of L1 VLP-based immunogens to target diverse cutaneous HPV types is hindered by the type-specific antibody response induced by the L1 conformational epitopes responsible for virus neutralization. Development of a multivalent VLP-based vaccine against cutaneous HPV types of clinical relevance for immunosuppressed individuals is ultimately a technical hurdle given the immense variety of genotypes to be covered. Conversely, the papillomavirus minor capsid protein L2 harbors highly conserved regions that can induce neutralizing responses against homologous and heterologous viral types45, which revealed significantly increased neutralizing antibody titers against all the five tested HPV types. Although generally considered as the gold standard for measuring functional antibody responses to papillomaviruses, the L1-PBNA does not closely mimic the early stages of infection that occur in vivo. These early infection stages are critical for the proteolytic processing of the viral capsid, which leads to the exposure of the cross-neutralization epitopes comprised within the N-terminal region of L2. Consequently, the L1-PBNA has a suboptimal sensitivity for the detection of L2-targeting neutralizing antibodies. Furthermore, the in vivo functionality of the neutralizing antibodies elicited by the cHPV vaccine leads was confirmed by their ability to afford partial or complete reduction of HPV-derived radiance signal via cervico-vaginal and cutaneous challenge in naïve mice, even for cHPV types whose neutralizing antibody titers measured with the L1-PBNA were rather low or barely detectable. Importantly, none of these in vitro neutralization assays capture protection afforded by antibody-dependent phagocytosis, a mechanism recently identified for L2 antibody-mediated responses46.Here, we demonstrated that our leading vaccine candidate, PfTrx-L2c12merOVX313, consistently induces neutralizing antibody responses against eight out of eleven tested cutaneous HPV types in mice. An even more favorable picture emerged from immunization studies in guinea pigs, with the induction of neutralizing antibodies against 19 (out of 21) tested cutaneous HPV types. It is also important to note that neutralizing antibody titers measured with the L1-PBNA likely underestimate the actual humoral responses induced by the cHPV vaccine prototypes. This view is supported by the results we obtained with the more sensitive FC-PBNA38. Oligomeric reformulation yielded monodipersed heptameric nanoparticles, which could be purified to high homogeneity by a thermal treatment (80 °C) followed by a single chromatographic step. We believe that oligomeric epitope display, plus the presence of additional T-helper epitopes associated to the OVX313 heptamerization domain, are responsible for the enhanced immunogenicity of the OVX313-conjugated antigens39. The modular nature of our approach was thus instrumental to the identification of what we believe is the best compromise between antigen complexity and multi-immunogenicity. For example, despite the inclusion of the OVX313 domain, the simpler PfTrx-L2-c6mer antigen elicited a fairly weak anti-HPV41 response—much lower than that afforded by the more complex PfTrx-L2c9merOVX313 antigen. In this regard, the HPV41 L2 epitope seemed essential for inducing type-specific neutralization in the context of our vaccine platform. Similar results were obtained in the case of HPV63, with an additional interesting aspect: cross-neutralizing antibody titers to HPV63 were likely induced by the HPV49 L2 epitope, only present in the PfTrx-L2c14merOVX313 antigen. Supporting our assumption is the fact that both L2 epitopes display two amino acids residues—Ala and Asn—at the corresponding position, in a very unique manner among all other epitopes explored. This highlights how a single epitope can critically contribute to the immune-performance of our modular vaccine design. However, the increased broadness of protection afforded by high-complexity antigens such as PfTrx-L2c14merOVX313 may come at the expense of lower recombinant protein expression levels and a less straightforward purification process. This kind of balance between immunogenicity and ease of production makes us envisage PfTrx-L2c12merOVX313, which can be obtained with a relatively high yield under GMP-compatible production conditions, as the leading candidate for a broad-coverage vaccine against cutaneous HPVs.Comprehensively strong immune responses against many cutaneous and even mucosal HPV types were induced by the monomeric PfTrx-L2c6mer and PfTrx-L2c9mer antigens in guinea pigs, but the same antigens proved to be surprisingly weak in mice. This yet unexplained immunogenicity gap was partially compensated by antigen reformulation through genetic fusion to the OVX313 oligomerization domain, which has been previously applied successfully to the PANHPVAX vaccine47, suggesting a protective role of cHPV-specific antibodies against cutaneous lesions. Vaccine-mediated prevention against experimentally induced skin infection caused by papillomavirus has been demonstrated in rabbits48, cattle49, and horses50. A substantial body of epidemiological data has linked cutaneous HPV infection to squamous cell carcinoma54, a neoplastic pathology that is particularly aggressive in transplanted patients56. The most promising evidence supporting a protective role of vaccination against papillomavirus-induced skin cancer was obtained by Vinzón et al.57. In this work, the authors demonstrated that Mastomys coucha mice—a rodent species that is naturally susceptible to the development of skin tumors caused by Mastomys natalensis papillomavirus (MnPV) infection—were protected against benign and malignant skin tumors by anti-MnPV L1 VLP vaccination, both under normal as well as immunosuppressive conditions. In line with these findings, and in keeping with the predicted broadness of protection afforded by our multiepitope cHPV vaccine design, we have recently found that immunization of virus-free M. coucha with PfTrx-L2c12merOVX313 elicits the production of neutralizing antibodies leading to protection against MnPV-induced cutaneous tumors . Despite this fairly strong circumstantial evidence, final proof that antibodies are preventive against HPV-induced skin cancer in healthy and immunocompromised individuals will only be obtained from clinical trials in humans.Seminal research on papillomavirus has demonstrated that relapses from warts causally associated to HPV1 are linked to decreased humoral immune responses58) within cells located in sun-exposed body sites. Cells thus transformed would no longer depend on cutaneous HPV infection for progression toward malignant lesions, a carcinogenesis model named ‘hit-and-run’15. This model has recently been challenged by Strickley et al.59, who proposed that the loss of T-cell immunity against commensal papillomaviruses, rather than a virus-induced pro-oncogenic effect, is the cause of increased risk to develop skin cancer. In our opinion, conclusive evidence for a causative involvement of cutaneous HPVs in skin cancer could only emerge from vaccination studies, assuming a preventive effect by the vaccines against tumor formation.It has been proposed that the carcinogenic role of cutaneous HPVs in skin cancer relies on the virus-promoted accumulation of ultra-violet-induced mutations in essential tumor suppressor genes  epitopes on the capsid surface of HPV3, but this approach was not successful with other HPV types. Although artificial, the use of ‘hybrid’ PSVs produced results well correlated with the expected immunogenicity of our vaccine candidates. Nevertheless, caution is needed in interpreting immunogenicity data related to these particular HPV types. A minor limitation refers to the lack of data on the durability of neutralizing antibody responses induced by our lead candidate. Nevertheless, we have demonstrated that neutralizing antibody levels induced by the PANHPVAX are measurable for up to 1-year after immunization60, and we suspect this may also be the case for our cutaneous candidate vaccines.The major limitations of our study are related to (i) the restricted number of cutaneous HPV types evaluated by the in vivo murine challenge model; (ii) the low production yields of the PfTrx-L2c14merOVX313 antigen; and (iii) the lack of a demonstrated vaccine-induced immune-reactivity against some cutaneous HPV types targeted by the antigens . The first limitation is mainly due to the technical difficulties in establishing consistently measurable PSV infections at cutaneous sites. However, the comprehensive number of cutaneous and mucosal HPV types tested with our PBNA panel partially compensates for this limitation. Regarding the second point, and in view of our goal to design a low-cost, broadly protective vaccine, we reasoned that despite its slightly lower performance compared to PfTrx-L2c14merOVX313, the immunogenicity profile displayed by the PfTrx-L2c12merOVX313 antigen meets the requirements of a broadly protective vaccine. The third limitation, namely the production of sufficiently active cutaneous HPV pseudovirions, has to do with a still unsolved technical challenge experienced by different laboratories24. The prevalence of skin warts also increases with time since transplantation61. Whether this is due to recrudescence or new HPV infections remains to be demonstrated. It should be noted, likewise, that the skin colonization by HPV is a very dynamic process, which shows distinct age- and sex-dependent seroprevalence patterns for phylogenetically distinct genera. Antibodies to cutaneous mu- and nu HPV types appear early in life whereas those to beta- and gamma cutaneous types are rare in children and accumulate later in life 62. Therefore, children, adolescents, young-, middle-aged and older adults electable for organ transplantation are all at higher risks of developing skin lesions, and may have different windows of opportunity to benefit from a vaccine targeting cutaneous types. What is of utmost importance, in our opinion, is the understanding that the value of a cutaneous HPV vaccination for preventing post-transplantation skin lesions can only be evaluated in clinical studies with OTRs of different ages, before the onset of immunosuppression, mainly due to the knowledge gaps and shortcomings on the role of cutaneous HPV in skin lesions. A similar vaccination protocol is already in place for other vaccines administered to OTRs such as measles, mumps, rubella, varicella, pneumococcus, influenza, hepatitis B, meningococcal and other vaccines63. Assuming a protective effect against skin lesions, vaccination protocols can be further adapted (i.e. as a routine in the childhood) according to the follow-up studies, as still happens for the currently available HPV vaccines targeting anogenital cancers . A vaccine specifically targeting cutaneous HPVs would thus represent an important prophylactic addition, but it could also shed light on the causative role of cHPVs in skin carcinogenesis.In conclusion, we document the development of a broadly immunogenic prototype vaccine against cutaneous HPV types associated to post-transplantation skin lesions in organ-transplanted patients. Our leading vaccine candidate, PfTrx-L2c12merOVX313, also appears to be well suited to a GMP-compatible production process—an additional important prerequisite for entering a clinical study. It should also be noted, in this regard, that PANHPVAX, the L2 polytope-based mucosal HPV vaccine that shares the same scaffold components (PfTrx and OVX313) as our lead cHPV vaccine candidate, will enter into a phase I clinical evaluation mid of 2022. Because of such compositional similarity, this phase I study will thus also provide indirect but important information on the safety, and prospective in-humans efficacy, of PfTrx-L2c12merOVX313. In this respect, PANHPVAX has been successfully manufactured under GMP and GMP protocols could be readily adapted to PfTrx-L2c12merOVX313. A prophylactic vaccine capable of protecting organ transplant recipients against HPV-related skin lesions should induce a broad-spectrum immune response. We propose a strategy of immunization prior to iatrogenic immunosuppression based on the observations that OTRs are at greater susceptibility risks to acquire skin lesions presenting multiple HPV types following transplantation37  were further C-terminally fused to the OVX313 oligomerization domain, a derivative of the complement inhibitor C4-binding protein65. Codon-optimized genes coding for eight antigens were chemically synthetized (GenScript) and genetically inserted into the NdeI-BamHI site of pET24b expression plasmid. The resulting constructs were verified by sequencing and transformed into electro-competent Escherichia coli BL21 codon plus (DE3) cells.Putative cross-neutralizing epitopes comprising 19 residues found in the N-terminus of the L2 protein of several cutaneous HPV types were predicted by sequence homology to the HPV16 L2 amino acid (aa) 20–38 epitope previously characterized by us66. The resulting translucent supernatant was carefully collected in order to avoid contamination by the opaque pellet formed by micelles pelleted down in the tube. The procedure was repeated once, and the final supernatant aliquots were then stored at −20 °C.Recombinant protein expression of the different antigens were typically assessed in small-scale (4 ml) overnight cultures (LB medium at 30 °C) in the presence of 2 mM IPTG. Upon confirmation of expression via electrophoresis in Coomassie-blue stained SDS-PAGE gels, protein production was scaled-up to 400 ml bacterial cultures, followed by cell harvesting and washing, and lysis via high pressure (French press) in the presence of 25 mM Tris, 300 mM NaCl, 0.1 mg/ml lysozyme, 1 mM PMSF, 0.16% Tween 20 at pH 8. Following centrifugation of bacterial total lysate, the supernatant (cleared lysate) generally underwent a heat treatment (incubation at 65–85 °C for 30 min) that yielded considerably pure (>80%) protein preparations, given the extreme thermo-stability of the PfTrx scaffold protein. For the OVX313-fused versions of the antigens, we developed an additional cation-exchange chromatography step through which the protein samples were loaded onto a HiTrap sepharose column  equilibrated in 0.2 M NaCl-containing PBS, pH 8. The target antigens were eluted via a saline gradient (0.2–1 M NaCl-contaning PBS buffer). Individual peak fractions monitored by SDS-PAGE were pooled, exchanged into PBS pH 7 and quantified via SDS-PAGE against a curve of standard BSA. The spontaneous oligomerization of OVX-fused antigens was evaluated by SDS-PAGE under non-reducing conditions, size-exclusion chromatography and dynamic light scattering as described below. The final protein samples were detoxified as follows: 1% (v/v) Triton X-114 was added to each protein prep and the mixture was incubated on ice for 5 min, and then for additional 5 min at 37 °C. Next, the samples were centrifuged at 10,600 rcf for 1 min at 37 °Chttps://www.dsmz.de/microorganisms/medium/pdf/DSMZ_Medium242.pdf) or in soya-supplemented BMM (SBMM).Production and purification of leading candidates was also assessed in proprietary minimal BMM , a MF59-like a squalene-based, oil-in-water nanoparticle emulsion afforded robust L2 neutralizing antibody responses when formulated with our PfTrx-L2-OVX313 prototype antigen targeting HPV1640. Therefore, we employed AddaVax as adjuvant for the cutaneous HPV candidate vaccines explored in this current pre-clinical study. All mice were immunized four times at biweekly intervals with intramuscular injections of 50 µl doses containing 20 µg of the antigens adjuvanted with 50% (v/v) AddaVax (InvivoGen). Four weeks after the last dose, the mice were sacrificed and blood samples obtained via heart puncture. 150–200 g outbread Hartley (Crl:HA) female guinea pigs   were immunized subcutaneously four times at triweekly intervals with 100 µl doses containing 30 µg of the antigens similarly adjuvanted with AddaVax. Collection of blood samples proceeded as described for mice.Six- to eight-weeks-old female BALB/c mice were purchased from Charles River Laboratories  and kept under specific-pathogen-free conditions . We have previously demonstrated that the adjuvantation of our candidate vaccines is necessary to induce strong L2 neutralizing antibody responses against mucosal HPV typeshttps://pave.niaid.nih.gov/) of 18 cutaneous HPV types were synthetized (GenScript) and cloned downstream of a CMV promoter . The L1-L2 bicistronic region contains an IRES sequence between the capsid genes, and a bovine growth hormone poly(A) tail. PSV constructs for HPV1, HPV2, and HPV41 were designed differently: the nucleotide sequence encoding the HPV3 L2 aa 1–46 was replaced by the corresponding HPV1, HPV2, and HPV41 L2 regions, giving rise to ‘hybrid’ HPV3 PSV displaying those grafted L2 aa20–38 epitopes. All PSV constructs designed for this work were confirmed by sequencing. The following PSV constructs were kindly provided by colleagues: p5shell from Dr. John Schiller ; p38shell and p76shell from Dr. Joakim Dillner ; p92shell and p96shell from Dr. Reinhard Kirnbauer . Nucleotide sequences of those constructs are available at https://ccrod.cancer.gov/confluence/display/LCOTF/Home.Cutaneous HPV pseudovirions (PSV) were used as virus surrogate during our in vitro neutralization assays and murine challenge models (Supplementary Table 6 cells) were seeded in 10 ml of DMEM supplemented medium per 10 cm tissue culture dish (10 dishes were used per PSV prep) and co-transfected 16 h later with 15 µg of a HPV-related construct plus a reporter plasmid coding for a Gaussia-luciferase protein (GL), using polyethylenimine . Cells were incubated for 3 days in a humidified incubator at 37 °C in the presence of PEI/DNA complexes. Transfection efficiency was monitored by fluorescence microscopy, as the GL-reporter plasmid also encodes a green-fluorescent protein (GFP). At the third day of transfection, cells were harvested from the culture dishes, transferred to 50 ml conical tubes and centrifuged for medium removal. After washing the cells in PBS and transfer to 2 ml low-binding tubes (Sigma-Aldrich), a new centrifugation took place in order to pellet the cells. For each milligram of cell pellet harvested, 1 µl of DPBS supplemented with 10% Brij58 (v/v) (Sigma-Aldrich) and 0.1% RNAse cocktail (v/v) (Thermo Fischer) was used to re-suspend the cells by inversion. Incubation of cell lysate proceeded for 2 days at 37 °C in an end-over-end rotator for maturation of PSV. Then, the lysate was chilled on ice for 5 min and salt concentration of cell lysate was adjusted to 850 mM by adding 0.17 volumes of 5 M NaCl. Following an additional incubation on ice for 10 minutes, the lysate was cleared by centrifugation  and transferred to a new low-binding tube placed on ice. DPBS/0.8 M NaCl was used to wash and extract PSV particles loosely bound to the pellet obtained after centrifugation of the lysate (1 µl of buffer per milligram of pellet). After centrifugation, both cleared lysates were combined and incubated with 2.5 units/ml turbo nuclease (Jena Bioscience) for 1 h at 37 °C.Human embryonic kidney cells , according to the instructions of the manufacturer. A microplate luminometer  was used to determine the culture medium associated luminescence after 15 min of substrate addition. Confirmation of PSV-derived transduction was achieved by a combination of polyclonal and monoclonal neutralizing antibodies. PSV infectivity was determined based on the fraction dilution yielding 100-fold higher relative luminescent units (RLU) as compared to its control in the presence of a neutralizing antibody.Purification of PSV particles from the cleared lysate was carried out by ultracentrifugation through a 27, 33, and 39% Optiprep step gradient for 5 h at 16 °C at 234,000 rcf at acceleration 9 and deceleration 1. After ultracentrifugation, 0.5 ml gradient fractions were collected in 1.5 ml low-binding tubes and screened for transduction activity in cell culture. For this, aliquots of each gradient fraction were diluted 1:1000 with DMEM supplemented medium and added to 12.5 × 10³ HeLa TK4 cells45. In brief, PSVs are subjected to maturation in the presence of furin, which proteolytic activity has been demonstrated to be a critical step for HPV infectivity. Furin cleaves L2 at its N-terminus allowing exposure of the targeted cross-neutralization epitopes and further binding to secondary cell receptors before infection. Following purification as described above, the transducibility of fc-PSV particles is then measured in furin-deficient LoVoT reporter cell line, which is permissive to fc-PSV transduction while insensitive to non-cleaved PSV ones.The production of furin-cleaved-(fc-) PSV was carried out in a furin-overexpressing 293TTF producer cellsFor the in vivo HPV challenge models, PSV particles carrying a firefly-luciferase (FFL)-encoding plasmid were produced and purified as described above. The intracellular FFL transduction was measured with Beetle-Juice luciferase assay , according to the manufacturer’s recommendations.68. Briefly, serum samples were serially diluted six times in a 96-well plate using a threefold increment to achieve a final serum dilution in the neutralization assay of 1:50 to 1:12,000. Next, the PSV previously diluted in DMEM were added to the diluted sera and the mix incubated for 20 min at room temperature. Finally, HeLa TK4 (106 cells) were added to the PSV-serum mixture and the assay plate incubated at 37 °C for 2 days in a humidified incubator. PSV-transduced GL activity was determined as described above. All sera were tested in duplicates. The serum neutralization activity (% inhibition) was calculated via the GraphPad software program by normalizing the luciferase signal measured for ‘PSV-serum’ against the corresponding signal for ‘PSV-alone’. Neutralizing antibody titers (EC50) represent the serum dilution in which a 50% inhibition of PSV-induced luciferase signal is seen. EC50 values <50 were considered non-neutralizing and therefore set to 0.1.Neutralizing antibody titers were determined by the standard PBNA 45 and employing fc-PSV particles was also deployed to complement our analysis. This assay is better suited to the detection of L2-raised neutralizing antibodies as a result of the improved display of targeted aa 20–38 cross-neutralization epitopes following furin cleavage of the HPV capsid. Neutralization of fc-PSV particles is assessed with the use of the furin-deficient LoVoT reporter cell line. The assay layout is similar to the L1-PBNA.An alternative assay named FC-PBNA38. For this, male mouse cage bedding was transferred to groups of female mice for inducing hormonal synchronization . Two days later, 3 mg of medroxyprogesterone acetate  were administered subcutaneously. On the following day, each mouse was injected intraperitonealy with 100 µl of a pool of immune sera . One day after passive transfer of sera (day 5), the mice were treated intravaginally with 50 µl of 4% nonoxynol-9  in 4% carboxymethyl cellulose . Four hours later on the same day, HPV PSV in 4% CMC was instilled intravaginally. The in vivo PSV-derived transduction was measured two days later by instilling 20 µl luciferin substrate  intravaginally and using an in vivo imaging system (IVIS) imager . A region-of-interest (ROI) around the luminescence emitting area of each mouse was obtained using the Living Image 2.50.1 software  to determine the average radiance . The background signal was obtained by imaging each group of mice prior to instillation of luciferin, and subtracted from the average radiance obtained post-instillation.Six to eight-week-old female BALB/c mice were obtained from Charles River Laboratory  and subjected to a cervico-vaginal PSV challengeFor the cutaneous PSV challenge, the animals had its dorsal hair removed with an electric clipper followed by application of a depilatory cream for 20 s. Under anaesthetic, an area of 2 × 1.5 cm of the shaved region was carefully scraped using a scalpel to remove the uppermost layers of the epidermis without causing cuts or bleeding. Two days later, 20 µl of cutaneous PSV in 0.6% CMC (1:2) was added to the scrapped region. The luminescence read-out was assessed 2 days post-infection by dropping 30 µl of luciferin substrate onto the scarified region. Determination of average radiance was carried out as described for the cervico-vaginal challenge model.A Superdex 200 Increase 10/300 GL column  equilibrated with PBS at a flow-rate of 0.5 ml/min was employed for size-exclusion chromatography (SEC). The column was applied in an Äkta fast protein liquid chromatography (FPLC) system, using UV 280 nm wave-length and 1.5 MPa as the maximum pressure, carried out with a Unicorn 5.0 software . Protein elutions were obtained with 2 column volumes of running buffer. Fractions of 1 ml were collected and stored at 4 °C until further analysis by electrophoresis in SDS-PAGE gel and coomassie dye-binding assay. A Zetasizer Nano ZM device  was employed for analysis of the hydrodynamic sizes of nanoparticle antigens by dynamic light scattering (DLS).HPV type-specific seroprevalence  with 95% confidence intervals (using the method of Clopper-Pearson), and mean neutralizing antibody titers with standard error of mean were calculated using GraphPad 8.3.1. Statistical significance was determined with the nonparametric Mann-Whitney test, also via GraphPad.Further information on research design is available in the Supplementary MaterialREPORTING SUMMARY


Despite low knowledge levels, there was high parental willingness to have their children vaccinated.Cervical cancer can be prevented by human papillomavirus (HPV) vaccination. However, parents can have concerns about vaccinating their daughters. Consequently, there is a need to identify prevalence and risk factors for HPV vaccine hesitancy among parents in Kenya. A descriptive cross-sectional study was conducted among parents with children aged 9–14 years attending a leading referral hospital in Kenya. Data on sociodemographic traits, HPV knowledge, beliefs and vaccine hesitancy were collected. Out of 195 participants, 183 (93.5%) were aged >30 years. Thirty-four (46.4%) of males and 39 (35.1%) of females did not know that the vaccine is given to prevent HPV infection. Encouragingly, levels of vaccine acceptance were high (90%) although one-third (37.9%) had a negative perception about the effectiveness of the vaccine, with vaccine hesitancy attributed to safety concerns (76%) and feelings that the child was too young (48%). Positive beliefs and knowledge of the vaccine were positively associated with parental willingness to vaccinate their children. Low levels of parenteral education and a younger age among mothers were negatively associated with willingness to vaccinate. Most parents (59%) would consult their daughters before vaccination, and 77% ( Cancer is the leading cause of death among women, especially in low-income countries , with ceIn LMICs, cervical cancer ranks fourth among all female cancers in terms of morbidity after breast cancer, colorectal, lung cancer in that order (estimates for 2020), with a ratio of mortality to incidence of 57% . In addiAmong the leading risk factors for cervical cancer is prolonged exposure to HPV . Early sThere is also a link between HIV infection and higher rates of HPV acquisition ,22. ThisPublished studies have shown that the HPV vaccine is safe and effective against cervical cancer ,26,27. TFollowing HPV vaccine development, the Global Alliance for Vaccines and Immunization (GAVI) supported vaccine demonstration projects in 2012 among LMICs. GAVI supported and co-funded 2-year HPV vaccine demonstration projects with a view to eventually planning and implementing nationwide HPV vaccination programs among countries in Sub-Saharan Africa as seen in Rwanda. Kenya received GAVI support , with thHowever, there can be concerns towards the HPV vaccine across countries, exacerbated by misinformation, impacting on its uptake ,32,33,34This was a descriptive cross-sectional study conducted among adults of either sex seeking care at the medical clinics of Kenyatta National Hospital (KNH), between June and August 2020. KNH was selected for this study as it is a leading public tertiary care hospital in the Capital City of Kenya, Nairobi, representing an urban population. KNH is also the largest referral facility in East and Central Africa , and mosSample size determination was calculated using the Cochran formula , as thisThe patient numbers attending the clinics during the COVID-19 pandemic reduced substantially; despite this, the numbers attending the clinic were large enough to achieve the desired sample size. The hospital had restricted attendance during the earlier days of the pandemic, but these restrictions were already lifted at the time of this study. Participants were eligible if they were aged above 18 years and were a parent to a pre- or adolescent child aged 9–14 years. In addition, they had to be able to communicate fluently either in Swahili or English, visited the medical clinic of KNH during the study period and provided informed written consent for participation. Women and men who self-reported that they had been previously diagnosed and treated for either cancer of the cervix, penile cancer or genital warts, were excluded from this study. These parents were excluded as by experiencing the disease, they would have better knowledge of cervical cancer and might be more receptive regarding the vaccine, leading to biased responses. As a result, further compromise the representativeness of the study findings.The hospital has eight outpatient pharmacies, each serving specific types of patients. One of them (Pharmacy 15) serves the medical clinics, which target patients with chronic comorbidities such as hypertension and diabetes. Medical clinics are held daily, each booking approximately 30 patients per day. Pharmacy 15 was identified as most suitable for recruitment of participants. This is because patients typically collect their medication from this pharmacy after consultation at the medical clinics. Most of the patients attending medical clinics are aged above 40 and therefore more likely to have adolescent children. Participants were approached and recruited as they collected their medicines from Pharmacy 15.We aimed to recruit 10 patients daily as it is a realistic number of participants that could be handled comfortably by the data collectors. Since approximately 30 patients are seen daily, a quasi-randomized sampling method was used. Hence, every third patient was approached when they presented themselves to collect their medication, aiming at the desired sample size of 10 participants per day. In the queue, participants were briefly informed about this study and invited to participate. Those who expressed interest in participating were invited to the Medicines Information Centre office, which is a private room located in the pharmacy for further screening and potential recruitment. They were subsequently screened more thoroughly for eligibility, and written informed consent was obtained for those willing to participate. On some days, the targeted sample size of 10 per day was not achieved because some of the invited potential participants did not meet the eligibility criteria or declined to participate during the screening process. Sampling and recruitment continued on a daily basis, until the desired sample size was achieved.Data were collected using a questionnaire administered in a face-to-face interview by the data collectors. The questionnaire was adopted from previous studies ,49, and The questionnaire was subsequently pretested on 20 study participants who met the eligibility criteria before the principal study was rolled out. The findings showed the instrument’s internal consistency was sufficient as indicated by a Cronbach’s alpha value of 0.78. Questions where there were concerns with comprehension were subsequently modified before commencement of actual data collection.TM version 7.2.4.0  database. The data were cleaned to ensure consistency in the format of the responses. Missing entries were identified, and an attempt was made to identify sources of error in data collection and rectify them as far as possible to ensure data completeness. Only the principal investigator had access to the documents, with the database password protected and backed-up daily to minimize accidental data loss. The database was locked at the end of data collection so that any future fraudulent entries could be tracked. All data are archived for 10 years in line with Kenyan law on archiving of research data.Within 24 h of data collection, responses were entered into an Epi InfoWe used three outcome variables, namely the score obtained on the knowledge regarding HPV infection and the HPV vaccine, beliefs about HPV vaccine score and their willingness (acceptance) to have their child vaccinated. The first two outcome variables were numeric and the last was dichotomous. The key predictor variable was parental sex. The other predictor variables included sociodemographic traits and specific aspects of knowledge.The knowledge score for each individual was obtained by calculating the sum of all the correct responses to the 30 questions that were designed to elicit information on knowledge on HPV infection, cervical cancer and the HPV vaccine. The maximum score was 30. The scores were subsequently converted to a dichotomous variable (0 for No or ‘I don’t know’ and 1 for Yes). A cut off value of greater than 15 for the 30 questions on knowledge was used to show greater knowledge, based on prior research.The belief score was obtained by the simple sum of the responses to the questions on attitudes and beliefs. The responses were categorized as negative beliefs (1), not sure (2), and positive beliefs (3). The maximum score was nine.Categorical variables were summarized as frequencies and percentages. The continuous variables were tested for normal distribution using the Shapiro–Wilk test and histograms were plotted to examine the distribution. Normally distributed variables were summarized as the mean with a standard deviation, and those not normally distributed were summarized as the median and interquartile range.Inferential analysis was conducted to compare the characteristics of parents who were willing to have their children vaccinated with those who were unwilling. Inferential tests used were the unpaired t-test for continuous variables, the Mann–Whitney test for variables not normally distributed and the chi square test for categorical variables.Because the knowledge and belief scores were not normally distributed and therefore generalized, additive regression modelling was used to identify the key determinants of knowledge.mgcv package in R was used for general additive modelling.Logistic regression was undertaken to identify factors that influence parental willingness to vaccinate their children. Multivariate regression controlled for confounding such as education levels. Covariates that were significant on bivariate inferential analysis were included in the regression models. The parsimonious models were identified using stepwise backward elimination. The level of significance was set at 0.05. Data cleaned in Epi Info were exported to STATA Version 13 and R 4.3 software for statistical analysis. The Approval to conduct this study was obtained from Kenyatta National Hospital—University of Nairobi (KNH/UoN) research and ethics review committee (Reference number: KNH-ERC/A/178). The Kenyatta National Hospital administration and Head of Department of the medical clinics granted permission to conduct this study. Voluntary informed consent was sought from all study participants before they started the questionnaire. Confidentiality and privacy were maintained by replacing identifier information with a code and database protection.We approached a total of 488 people. Out of these, 147 (30%) declined to participate in this study. The remainder, 341 (70%), were screened for eligibility, of whom 195 (57%) met the inclusion criteria and agreed to participate. Most parents recruited into this study were aged above 30 years (93.5%), with more females than males and most being self-employed. Secondary education was the highest level of attainment for most participants . Nearly all participants were married  and the majority of participants had children aged between 12 and 14 years. Other than three participants, all were Christians.We did not collect data on income since the HPV vaccine is provided free of charge through donor programs. More details on the baseline sociodemographic characteristics of the parents are summarized in The histogram summarizing the performance of the participants, with a maximum value of 30, is presented in p < 0.001). Data transformations were applied to make the variable conform to the normal distribution; however, these transformations were not successful.p = 0.025). The findings on the sources of information on the HPV vaccine are summarized in The most common source of information on the HPV vaccine was the participants’ fellow workers , while religious leaders were the least common source of information. Women had a greater variety of information sources compared to men, with more females obtaining their information from health care providers (p = 0.040). Of the female participants, 64.9% (72) accurately stated that the vaccine is for the prevention of HPV infection. For both males and females, less than 36% knew that the vaccine is useful for the prevention of genital warts; in this aspect, males performed better than females. More women (75.5%) than men (57.1%) knew that young adults could also be vaccinated (p = 0.021). Of concern is that there were misleading beliefs with 40.1% of participants falsely thinking there was no need for a Pap smear after getting the vaccine. Worryingly, many participants could also not link the virus to cervical cancer and only slightly more than 50% had heard of HPV infection. Overall, 60% of women were aware that HPV is linked to cervical cancer as opposed to only 41.7% of male participants (p = 0.035). Generally, there was poor knowledge about the mode of transmission, risk factors, and symptoms, with less than 40% of participants who responded correctly to these questions.More females  compared to males  knew that the HPV vaccine was available free of charge for adolescent girls. In addition, 71.2% of participating females knew the vaccine was for the prevention of cervical cancer (p < 0.001). Encouragingly, approximately 80% of participants knew that cervical cancer is preventable.Generally, there was good knowledge about cervical cancer with almost all participants having heard about cervical cancer (95%). With regard to knowledge on cervical cancer and HPV infection, there were no statistically significant differences in the numbers of correct responses among males and females. Unfortunately, though, few male parents knew that cervical cancer could be diagnosed using the Pap smear test . Both sexes (77%) would recommend the vaccine to a 10-year-old.More females (65%) than males believed that the vaccine is effective; however, there were no statistically significant differences across the sexes. More women (71%) than men (64%) considered the vaccine to be safe  had the highest score .Knowledge was the strongest determinant of the belief score. There was a negative association between having adequate knowledge and beliefs . This means that increasing knowledge about HPV may not necessarily translate into positive beliefs. Education status had no association with the belief score.Both males and females showed a high level of willingness (90%) to vaccinate their children. Reasons given for the willingness to have their child vaccinated included positive peer pressure (52%); request by the adolescent (60%); government and school’s recommendation (68%); and doctor’s recommendations (86%). Overall, 94% of the participating female parents were willing to accept the HPV vaccine because of its effectiveness compared to 85% among their male counterparts. More than 70% of parents felt that they did not have adequate information for decision making concerning vaccinating their children despite generally positive attitudes. None of the males were sympathetic about pain at the injection site and less than 26% of parents would reject the vaccine because of their child’s refusal. Encouragingly, less than 4% of the women and 1% of men were against all vaccinations. Similarly, across both males and females, more than 74% would reject the vaccine due to HPV vaccine-related safety concerns if these occurred. Short-term side effects were considered a hindrance by 45% or more of parents. Nearly half the parents felt that their daughters were too young to be vaccinated.Similarly, 25% or more of parents would reject the vaccine because of concerns that their daughters would be stigmatized due to accusations of being promiscuous after being vaccinated. Of note was that 16% of females and 13% of males would reject the vaccine because their religion does not allow vaccination. In addition, more females than males considered the vaccine to be unnecessary. More females than males would also reject the vaccine for the children if they were to incur any vaccine-related cost such as transportation to the health facility. All reasons identified for acceptance are summarized in There was a positive association between willingness to vaccinate their child and beliefs  and between willingness and knowledge levels . After adjusting for confounding variables with the knowledge score and the beliefs score, males were more willing than females to have their children vaccinated even though they had less knowledge; however, the measure of association was not statistically significant . This means that men who had similar levels of knowledge as women would have been more willing to have their children vaccinated.With regard to sex, qualitative confounding was observed in that the crude measure of association showed a negative association between sex and willingness to have the child vaccinated. After adjusting for confounding, the association increased in magnitude and direction. The crude measure of association was confounded by knowledge.p = 0.040). In addition, there was a negative association between parental education level and willingness to vaccinate. The higher the education level, the lower the willingness to vaccinate their children. Level of occupation and marital status had no association with willingness to vaccinate their children.There was a significant negative association between age and willingness to have their child vaccinated  a notable reason. This is similar other studies in Kenya revealing safety concerns ,17,29,30A key concern was that the child was too young to be vaccinated with the HPV vaccine, which has been reported in other studies , with soThis is important as our study found a negative association between parental level of education with willingness to vaccinate. These negative associations were significant even after adjusting for beliefs and knowledge. Similar findings were seen in Thailand, although this was not statistically significant , althougWe also found a negative association between older parents and their willingness to have their children vaccinated. This differs though from findings in Thailand and the United States ,73. ConsMost participants in our study had heard about HPV from their colleagues at work. Health officers and television were also mentioned as a significant source of health information. This differs from findings in the United States amongst African Americans which found that medical professionals were the most important source of information for parents, followed by the television .Another concern was the general poor knowledge about HPV, and its association with cervical cancer, and the availability of the HPV vaccine among both male and female participants, similar to a previous study in Kenya study  and a syFinally, another key concern was that a significant portion of the study population held incorrect beliefs with only 60% of respondents believing that the vaccine was effective. However, this was higher than among adult women in the USA, where only 29.8% believe that the HPV vaccine effectively prevents cervical cancer .Overall, our findings highlight the need for a multipronged approach to improve future HPV vaccination rates in Kenya, similar to the situation in Rwanda ,15,16. TWe are aware of several limitations of this study. Firstly, this study was conducted in an urban area where parents were well educated and had better access to the HPV vaccine and information. Consequently, the findings may not be extrapolated to rural settings in Kenya. Secondly, we are aware that the sample was taken from an outpatient clinic and not a general population, which might reflect healthy behavior seekers. In addition, we did not explore the effects of religious dominations and culture on parental decisions as nearly all the participants were Christians. This is important given the influence of religious leaders in Kenya on vaccine usage ,39.Cultural aspects, such as limited decision making by women and stigma associated with reproductive issues, were also not adequately explored. Variables affecting vaccine access such as distance to the nearest public facility, knowledge about where the vaccine can be obtained, and the costs of travel to clinics were also not explored. There may also exist provider-related barriers to access to vaccines such as responsiveness, which were not addressed. However, despite these limitations we believe our findings are robust providing direction to the authorities in Kenya on ways to reduce vaccine hesitancy where this exists. We will be following this up in future research projects building on the suggestions arising from our research.The responses to questions on attitude and beliefs should also have been subjected to item analysis so that the overall score should have been weighted to levels of difficulty. This is an area for future research.Despite the low levels of knowledge and safety concerns, there was high parental willingness to have their children vaccinated. The key reason for vaccine hesitancy was insufficient knowledge and safety concerns. Men generally had lower levels of knowledge regarding the HPV vaccine compared to females. Key areas need to be addressed going forward given the low vaccination rates that existed in Kenya in 2020.


HPV-associated head and neck squamous cell carcinoma (HNSCC) is a cancer entity with unique biological and clinical characteristics that requires more personalized treatment strategies. As the backbone of conventional therapeutics, radiation is now harnessed to synergize with immunotherapy in multiple malignancies. Accumulating preclinical and clinical data have suggested the potential of radioimmunotherapy in eliciting local and systemic anti-tumor response via direct killing of tumor cells and immunogenic cell death. However, this effect remains uncertain in HPV-associated HNSCC. Owing to its intrinsic radiosensitivity and distinct tumor microenvironment, HPV-associated HNSCC may represent a good candidate for radioimmunotherapy. In this review, we provide a detailed illustration of the biology, the genomic features, and immune landscapes of HPV-associated HNSCC that support the synergism between radiation and immune agents. The interaction between radiotherapy and immunotherapy is described. We also highlight the present evidence as well as ongoing trials using different combination strategies in the recurrent/metastatic or definitive settings. In addition, we have summarized the challenges and outlook for future trial design, with special emphasis on radiotherapy optimization and novel therapeutic options to incorporate. With the global prevalence of human papillomavirus (HPV) as a most common sexually transmitted infection, the incidence of HPV-associated head and neck squamous cell carcinoma (HNSCC), particularly oropharyngeal carcinoma (OPC), has dramatically increased over the past decades. Currently, HPV-associated OPC accounts for 33% of all cases globally, with a highest prevalence reported in Lebanon 85%) and Sweden (70%) ,2. Radio% and SweClinically, HPV-associated HNSCC is featured by a proclivity towards male, younger age of onset, less consumption of tobacco and alcohol, specific anatomic site in oropharynx, and a histology of non-keratinized squamous cell carcinoma . As a diHigh-risk HPV strains, predominantly HPV16, represent the major driving factors that mediate the oncogenesis of HPV-associated HNSCC. Despite lacking direct evidence of precancerous lesions induced by HPV infection, HPV-associated HNSCC possibly resembles HPV-driven ano-genital cancers in their development that involves progression from normal epithelium to dysplasia, carcinoma in situ and eventually invasive cancer ,12. In lDue to the common smoking history, HNSCC presents a relatively high tumor mutational burden (TMB) that ranks in the upper quartile among different cancer types . AccordiHPV-associated HNSCC, primarily OPC, also exhibits unique immune microenvironment that harbors more pro-immunological features. Compared to its HPV-negative counterparts, HPV-positive HNSCC displays a T-cell-inflamed phenotype with increased CD4+ T-cells, CD8+ T-cells and cytokine production, as well as higher diversity of T-cell receptor repertoire ,23,24. TFollowing T-cell activation, HPV-associated HNSCC develops a negative feedback loop to forge an immunosuppressive microenvironment with upregulated expression of CTLA4 , CD39 anAs discussed above, the genomic and immunologic features of HPV-associated HNSCC support the rationale of immunomodulatory agents, ICIs that block PD-1, PD-L1 or CTLA-4 in this population of patients. Given the pre-existing tumor-specific immune cells in HPV-positive tissues, the ICIs can effectively reverse the immunosuppressive microenvironment by releasing the “brake” and reactivating effector T-cells, thus reinvigorating the anti-tumor response. p = 0.01]  . When ap = 0.01] . These d = 0.01] ,35,36.p = 0.01) and chemoradiation  when compared to HPV-negative disease [HPV-associated HNSCC is generally believed to be more sensitive to conventional chemotherapy and radiotherapy (RT). Direct evidence came from a trial by Fakhry et al., in which patients with HPV-positive HNSCC had higher ORR to induction chemotherapy  . In biopConversely, radiation can exert strong immunomodulatory effects on tumor microenvironment, which is regarded as the 6th “R” in radiobiology . MultiplTaken together, the interplay of the immune response and radiotherapy warrants the application of radioimmunotherapy in malignant tumors, even those traditionally deemed with a “cold” tumor microenvironment. HPV-positive HNSCC, with its natural advantages in radiosensitivity and immune-sensitivity, might represent a good candidate for radioimmunotherapy to maximize the therapeutic ratio.Given its compelling immunomodulatory effects, radiotherapy has been applied in a metastatic setting in multiple malignancies to elicit systemic immune response against tumor, or putative “abscopal effect”, while attaining proper local control. As was described by R.H. Mole in 1953, an abscopal effect refers to a phenomenon whereby localized RT induced a tumor shrinkage in distant sites that were not exposed to irradiation . Howeverp = 0.86). When regarding the virus status of HPV or Epstein–Barr virus (EBV), subgroup analyses in the SBRT plus nivolumab arm found a numerically but not statistically superior response in virus-negative patients compared to that in virus-positive cases  [In HNSCC, however, a phase II trial (NCT02684253) from Memorial Sloan Kettering (MSK) Cancer Center failed to replicate the ORR advantage with the addition of single-lesion SBRT (9 Gy for 3 doses) to nivolumab  . These rAlthough generally recognized with favorable prognosis, non-metastatic HPV-associated HNSCC is in fact a heterogenous entity with wide diversity of therapeutic effect and survival risk ,68. As mThe JAVELIN Head and Neck 100 trial (NCT02952586) was the first randomized phase 3 study to combine an ICI with CRT in high-risk locoregionally-advanced HNSCC (LA-HNSCC). With the inclusion of HPV-positive OPC  and HPV-positive non-OPC, as well as HPV-negative HNSCC (stage III-IVb), this trial was designed to evaluate the PFS benefit of concurrent and adjuvant avelumab based on standard CRT. However, the trial was terminated early due to the failure to reach its primary endpoint. Although with fair tolerability, avelumab exerted no improvement in PFS or OS, either in the whole population, the HPV-positive or HPV-negative subgroups. A non-significant trend towards higher PFS was observed only in those with a high PD-L1 expression . Comparep = 0.0118) and satisfactory outcomes . KEYNOTE-412 expanded this design into a phase III randomized trial. With enrollment of LA-HNSCC including p16-positive OPC (T4 or N3), KEYNOTE-412 was intended to answer the question whether escalating treatment intensity by adding pembrolizumab to CRT will translate into survival benefits eventually. The preliminary results will be released later in 2022.A phase Ib trial (NCT02586207) reported the safety and efficacy of concurrent and adjuvant pembrolizumab in conjunction to CRT  in LA-HNThe ECOG ACRIN EA3161 (NCT03811015) is a phase II/III trial that aimed to investigate the survival benefit of maintenance nivolumab following definitive chemoradiation in PFS and OS amongst high-risk HPV-positive OPC, including those with more than 10 pack-years of smoking history and advanced stage of T1-2N2-N3 or T3-4N0-3. Another phase III trial, IMvoke010 (NCT03452137), randomized high-risk LA-HNSCC including stage III HPV-positive OPC to receive adjuvant atezolizumab after local radiotherapy or surgery. As the patient population in both trials features a high tendency of local progression and distant relapse, the addition of adjuvant ICI might hopefully help eradicate residual or micro-metastatic cancer clones.The IMMUNEBOOST (NCT03838263) trial introduced two cycles of neoadjuvant nivolumab to CRTs in high-risk HPV-positive OPC, also selected based on tumor stage  and smoking history (>10 pack-years). The primary endpoint is the completion rate of full treatment. Interim analysis presented at the 2021 ASCO meeting showed the feasibility of this protocol, and the trial is continuing , awaitinA series of clinical trials assessed the feasibility of replacing concurrent standard-of-care agents with ICIs during radiotherapy in HPV-associated HNSCC. Single-agent ICI was used in the PembroRad trial, KEYCHAIN trial (NCT03383094), NRG-HN004 (NCT03258554), NRG-HN005 (NCT03952585) and CITHARE trial (NCT03623646) with similar design. The PembroRad trial has completed enrollment and the preliminary results were reported at the ESMO 2020 annual meeting. After enrolling 60% of patients with OPC (46% p16+), this trial found RT-pembrolizumab with less grade ≥ 3 acute toxicities but disappointingly no outcome improvement over RT-cetuximab , which hThe REACH study (NCT02999087) compared dual-agents of avelumab and cetuximab with cisplatin  in cisplatin-fit cohort and with cetuximab in cisplatin-unfit cohort among LA-HNSCC patients receiving definitive RT. With the inclusion of 61% cases with OPC (35% p16+), the REACH study showed a significantly lower rate of distant failure with RT-avelumab-cetuximab in the cisplatin-unfit cohort, suggesting the effect of ICI in eliminating potential micro-metastasis . NCT0316The NCT03715946 trial was designed to de-escalate the standard post-operative CRT by introducing ICI in p16-positive OPC with intermediate-high risk. Using concurrent and adjuvant nivolumab, the investigators expected to safely omit concurrent cisplatin and meanwhile reduce the RT dose to 45–50 Gy in 25 daily fractions.Another important scenario for radioimmunotherapy-based de-escalation would be the combination of ICIs and induction chemotherapy to select patients for subsequent de-intensified RT. The OPTIMA-II NCT03107182) is a phase II trial proposed on the heels of OPTIMA, which reported favorable outcomes and reduced toxicities in HPV-positive OPC, based on induction chemotherapy with carboplatin/nab-paclitaxel and ensuing de-intensified RT  is a pha. The CheWhen applied prior to surgery, ICI alone is limited to induce satisfactory complete (pCR) or major (MPR) pathologic response in HNSCC including HPV-associated diseases ,86. ReceIn general, radiation synergize with immunotherapy largely in a dose-dependent manner. Ablative RT, typically given with fractional dose >10 Gy for 1 to 5 fractions, mainly provides local tumor control via ICD-mediated mechanisms . HoweverThe fundamentally differing effect of high-dose and low dose- RT on immune response has led to the development of a new technique of “RadScopal” that integrates high-dose RT to generate “in situ vaccination” and low-dose RT to reverse stroma immunosuppression  and thusAccumulating evidence has supported the feasibility of multi-site irradiation in a metastatic setting. As was illustrated in detail by Chang et al. , multi-s3 metastases followed by pembrolizumab resulted in a local control comparable to that targeting the entire metastatic volume [Partial irradiation of metastases has also been proposed, based on preclinical studies that showed non-inferiority of irradiation to 50% of the volume of the tumor to that of the full volume. In the phase I NRG-BR001 trial that enrolled 79 metastatic patients including four with HNSCC, Luke et al. reported that partial SBRT to > 65 cmc volume . This reElective nodal irradiation (ENI), usually known as the prophylactic delivery of a radiation dose to the clinically uninvolved but at-risk nodal areas, targets subclinical metastases in draining lymph nodes (DLN) and is routinely used in HNSCC in the definitive setting, including HPV-associated HNSCC. However, with the growing importance of DLN as a major location of T-cell priming, larger-field ENI might hinder this process by directly killing CD8+ T-cells. In fact, ENI has failed to improve outcomes in multiple malignancies including esophageal squamous cell carcinoma , early-sTiming of ICIs to irradiation is of essential importance to exert a synergistic efficacy. According to Dovedi et al., effective anti-tumor response and long-term tumor control can be acquired only when PD-L1 blockade was administered concurrently with RT . RetrospTheoretically, any agent that acts against the intrinsic resistance of HNSCC might synergize with radioimmunotherapy to expand the therapeutic ratio. Based on the potential mechanisms of resistance, the combinational strategy could involve several aspects: (1) chemotherapy  that indOf note, the unique biological features of HPV-associated HNSCC could provide insight into disease-specific strategies. For instance, DNA immunotherapy targeting E6/E7 stimulated potent anti-tumor immune responses in a phase Ib/II trial , whetherIn summary, current evidence indicates that HPV-associated HNSCC has higher sensitivity to RT and immunotherapy. Given the synergetic effect of RT and ICIs, HPV-associated HNSCC may represent a favorable candidate for radioimmunotherapy due to its special biological, genomic and immunologic landscape. Preliminary studies, although rare, have indicated the tolerance and efficacy of radioimmunotherapy in HPV-associated HNSCC. Ongoing trials will further provide valuable insight into the potential of radioimmunotherapy in triggering abscopal effect in the metastatic setting and facilitating personalized treatment in the definitive setting, as well as the safety profiles in HPV-associated HNSCC. There are open questions regarding the optimal RT scheduling, volume, and sites to irradiate and the combinational agents to elicit the synergetic effect of RT and immunotherapy, future investigations in these fields should be encouraged.


In live streaming shopping, exploring the influencing mechanism of consumers’ participation is an important prerequi site for understanding consumer behavior in social commerce activities. The purpose of this study is to explore the relationship betw een technological and social factors  in live streaming shop ping and consumers’ purchase intention. The mediating roles of attraction and cognitive assimilation were also examined.This study collected 425 pieces of data through questionnaire survey. The structural equation model is established based on S-O-R frame. The hypothesis is tested by structural equation model.Our study found that that real-time interactive and media richness positively affect attraction; visibility, guidance shopping and media richness positive affect cognitive assimilation; cognitive assimilation and attraction positive affect consumers’ purchase intention; cognitive assimilation and attraction play a partial mediating role in the influence of technology and social factors on cons umers’ purchase intention of live streaming shopping.From the perspective of socio-technical, this study explores the influence mechanism of different influencing factors on consumers’ purchase intention in live streaming shopping. This study expands the application of IT affordance theory in the context of live streaming shopping, and reveals the mediating role of attraction and cognitive assimilation between social, technological factors and consumers’ purchase intention. AccordiCompared with traditional e-commerce, live streaming shopping has developed into a unique business model with its own distinct features. First, real-time interaction between buyers and sellers can greatly enhance information transparency. Customers can interact with the streamer in real time through barrage questioning during any live streaming shopping period, it not only can shorten the perceived distance between buyers and sellers but also can effectively reduce customers’ uncertainty about products, as well as greatly eliminate customers’ perceived risks caused by online virtual context. As a result, it can increase the trust extent between buyers and sellers . Second,The practice of live streaming shopping is increasingly popular; however, research on this streaming is still in its early stage. Previous studies paid limited attention to exploring the impact mechanism of live streaming on users’ purchase intention . WongkitAgainst the backdrop of these issues, we argue that there is a need to understand the contextualized factors driving consumers to make a purchase in live streaming and explore the specific impact mechanism that how these factors affect users’ purchase intention. To achieve this goal, we draw literature on social commerce and IT affordance and adopt a sociotechnical perspective to construct a contextualized model for the following three reasons. First, live streaming shopping is under the scope of social commerce, which is a prevalent socio-technical phenomenon, and the socio-technical perspective provides us with the opportunity to capture both social and technical elements simultaneously in framing and investigating technology-related societal issues . Second,The rest of this study is structured as follows. First, we review the related literature and provide a theoretical foundation. Then, based on the Stimulus-Organism-Response (S-O-R) framework, we build our research model from three levels: live streaming as a technology platform, live streaming as an online social context, and interplay of the technological functions and the social situation of live streaming. Next, we describe our research design and results. Finally, we discuss the study’s contributions, implications, and limitations, as well as future research. Our work is expected to shed light on theory development in this emerging live streaming research area and help practitioners understand how the desirable socio-technical system shapes consumers’ live streaming purchase intention.2.2.1.Live streaming shopping represents the completion of mobile payment and online trading activities, while e-commerce activities perform trading through the network of live streaming platforms . It main2.2.The Stimulus-Organism-Response model (S-O-R) includes three parts: stimuli from the internal and external environment, the state of the organism, and their behavioral responses . There aIn the research on live streaming shopping, researchers apply the S-O-R model to explore the relationship between stimuli in the live streaming environment, consumers’ cognitive and emotional states, and resulting behaviors. For example, 2.3.The affordance theory originated from the field of ecological psychology. It describes the phenomenon that the subject produces a certain behavior based on the perception of environmental characteristics . This thOver the recent few years, the affordance term has been widely used in the study of social commerce. Some scholars have studied the relationship building between buyers and sellers from the perspective of IT affordance, and the research results show that the functions of social media greatly affect the formation of strong and weak relationships between buyers and sellers . Differe2.4.In order to study the influence of technical and environmental characteristics on consumers’ purchasing behavior in live streaming shopping, we introduce a social-technology interaction perspective. Socio-technical interaction perspective holds that the system consists of two parts: social environment and technical characteristics . Social 3.3.1.Live streaming shopping has changed the way of information exchange in online shopping. The application of live streaming technology combines social media functions with online shopping, and streamers and audiences can exchange information through real-time interaction . The aud3.1.1.Real-time interaction refers to the possibility of timely information exchange between buyers and sellers . This kiH1a: Real-time interactivity has a positive impact on attraction.H1b: Real-time interactivity has a positive impact on cognitive assimilation.3.2.The mode of live broadcast shopping is realized with certain technical support. First, without the technology of information production and dissemination, such as live streaming, the audience would not be able to interact with the streamers or obtain product information through live streaming. Second, the streamers provide opinions on product selection through live streaming. The audience raises their questions and purchase demands through bullet screens, and the streamers provide answers and professional opinions through live streaming. Finally, live streaming shopping incorporates text, pictures, voice, video, and other social media information. Live streaming shopping not only realizes the function of online shopping but also realizes the function of social media. This study chooses visibility, guidance shopping, and media richness as the technical characteristics of live streaming shopping.3.2.1.Guidance shopping refers to the possibility of providing consumers with appropriate product choices or services based on their needs and preferences . In liveH2a: Guidance shopping has a positive impact on attraction.H2b: Guidance shopping has a positive impact on cognitive assimilation.3.2.2.Visibility is defined as the degree of information display about products, including text, pictures, videos, and other information visible to consumers . ConsumeH3a: Visibility has a positive impact on attraction.H3b: Visibility has a positive impact on cognitive assimilation.3.2.3.The concept of media richness originates from the theory of information richness theory, which is defined as the ability of information to change users’ understanding and communication through media communication . InformaH4a: Media richness has a positive impact on attraction.H4b: Media richness has a positive impact on cognitive assimilation.3.3.In the application of the socio-technical perspective, users’ cognition, emotion, and behavior are the main research directions. Users’ emotional tendencies and cognitive attitudes are the two main aspects affecting users’ behavior . Users’ Some scholars defined consumers’ activities from three aspects: cognition, emotion, and behavior . ConsumeH5: Attraction has a positive impact on purchase intention.H6: Cognitive assimilation has a positive impact on purchase intention.3.4.Previous studies have found that technology and social factors have an impact on consumers’ purchase intention, and this impact is realized by attraction and cognitive assimilation . AttractIn the context of live streaming shopping, the influence of technological factors and social factors on consumers’ purchasing behavior is usually realized through attraction and cognitive assimilation . AttractH7: Attraction mediates the relationship between real-time interaction, guidance shopping, visibility, media richness, and purchase intention.H8: Cognitive assimilation mediates the relationship between real-time interaction, guidance shopping, visibility, media richness, and purchase intention.In order to test the structural model and reduce the influence of non-study variables on the data analysis, our study included eight control variables. The control variables were gender, age, education background, monthly income, whether they knew about live streaming shopping, the time they used live streaming shopping, the number of times they used live streaming shopping, and the live streaming software they used.In the context of live streaming shopping, technical factors have an impact on live streaming shopping behavior through information technology. Based on the stimulus-organism-response (S-O-R) framework, from the perspective of socio-technology interaction, we construct a theoretical model from three levels: live streaming as a technical platform, the interaction between live streaming technical function and social context, and live streaming as an online social environment. The technical factors mainly include visibility, guidance shopping, and media richness, and the social-technology interaction factors mainly include real-time interaction. The user’s perceived attraction and cognitive assimilation are the internal organism state, and the purchase intention of live streaming is the response. 4.4.1.This study mainly adopts a survey for data collection. In order to ensure the content validity and measurement validity of constructs, the measurement items of this study are all adapted from the existing literature. By combining the situation of live streaming shopping, we designed a questionnaire that fits our study. In this study, we use a seven-point Likert Scale to measure the focus constructs. This study involved seven constructs. The measurement items of real-time interaction (RI), visibility (VI), purchase intention (PI), and guidance shopping (GS) were adapted from When collecting survey data, we published the questionnaire on China’s largest questionnaire platform “Sojump” and spread the link through the social platforms WeChat and QQ. We prepared random lucky money for the respondents to encourage them to engage. A total of 425 questionnaires were collected from 9 April 2021 to 25 May 2021. In addition, we set the screening question “Have you ever had a live streaming shopping experience” to screen the survey subjects. A total number of 223 valid questionnaires were obtained after excluding invalid questionnaires. The descriptive statistics of the sample are shown in 4.2.The selection of data analysis methods has an important impact on the reliability of research results. Because the measurement items used in this study are mostly from the existing literature. Due to the different research situations, it is necessary to ensure the effectiveness of the measurement items used in the live streaming situation concerned in our study. Therefore, this study first used SPSS25.0 software for exploratory factor analysis, deleted invalid load factor items (including error factor load and double factor load), and finally got the formal scale used in this study see . In the 4.2.1.In order to ensure that the measurement model can properly reflect the observed variables in this study, exploratory factor analysis was used to evaluate the validity and reliability. The main parameters include Cronbach’s α and composite reliability CR; . The valThe convergent validity of variables is evaluated by testing factor loading and average variance extracted AVE; . The fac4.2.2.Common method bias refers to the artificial covariation between predictor and outcome variables caused by the same data source or raters, the same measurement environment, the context of the survey variable, and the variable itself . This st4.2.3.R2 represents the extent to which structural models account for variations in variables , indicating that real-time interaction has a positive impact on attraction. The hypothesis of H1a is supported. The path influence coefficient of real-time interaction on cognitive assimilation is −0.015 (p > 0.1), indicating that the influence of real-time interaction on cognitive assimilation is not significant, and that hypothesis of H1b is not supported. The path coefficients of guidance shopping on attraction and cognitive assimilation were 0.061 (p > 0.1) and 0.121 (p < 0.05), respectively. The results indicate that guidance shopping had no significant effect on attraction, and the conclusion did not support the hypothesis of H2a. It also shows that guidance shopping has a significant effect on cognitive assimilation, which supports the hypothesis of H2b. The effect of visibility on attraction was not significant . Visibility had a significant effect on cognitive assimilation . It can be seen that visibility has no positive effect on attraction, hypothesis H3a is not supported. Visibility has a positive effect on cognitive assimilation, hypothesis H3b is supported. The path influence coefficients of media richness on attractiveness and cognitive assimilation are 0.401 (p < 0.05) and 0.373 (p < 0.05), indicating that media richness has a significantly positive impact on the attractiveness and cognitive assimilation. Hypotheses H4a and H4b are supported.From the above analysis, it can be seen that the path coefficient of real-time interaction on attraction is 0.188 (p < 0.001), which indicates that attraction has a significant positive impact on consumers’ purchase intention, and hypothesis H5 is supported. The path coefficient of cognitive assimilation on consumers’ purchase intention was 0.301 (p < 0.05), which indicated that cognitive assimilation had a positive effect on consumers’ purchase intention, hypothesis H6 is supported. The influence of attraction and cognitive assimilation on purchase intention is significant, and the results of statistical analysis are shown in In the relationship between attraction and consumers’ purchase intention, the path coefficient of attraction to consumers’ purchase intention is 0.648 (In this study, process plug-in in SPSS was used to test the mediating effect of the structural equation model within a 95% confidence interval as shown in The indirect effects of real-time interaction, guidance shopping, visibility, and media richness on consumers’ purchase intention through attraction and cognitive assimilation are not included in the 95% confidence interval, which indicates that there is a significant mediating effect in the structural model. Hypotheses H7 and H8 are supported.In order to test whether attraction and cognitive assimilation play a partial mediation effect or a complete mediation effect, our study uses 5.5.1.From the perspective of socio-technical, this study establishes a theoretical model and conducts empirical tests to explore the influence of social and technological factors on consumers’ purchase intention in live streaming shopping. Through testing the research hypothesis, we got a relevant conclusion.First, real-time interaction has a positive impact on attractiveness but has no significant impact on cognitive assimilation. Real-time interaction is an effective way to establish an emotional connection between buyers and sellers. At the same time, real-time interaction can strength the relationship between buyers and sellers. In the live streaming situation, the good interaction between the streamer and the audience is an effective way to enhance the attraction of live streaming shopping to consumers. The streamer reduces the consumers’ strangeness through self-introduction and product display; it can also close the psychological distance between them. The social connection between buyers and sellers through real-time interaction is a kind of emotional connection between the two sides, which is more stable and reliable than an ordinary business relationship. This study analyzes the particularity of live streaming shopping mode. Our research finds that the product information presented in the form of live streaming can attract consumers’ attention. In live streaming shopping, the impact of real-time interaction on cognitive assimilation is not significant. We believed that there are several possible reasons for this. First, in live streaming shopping, consumers will interact with other viewers while interacting with streamers, and information from different sources may produce conflict; the amount of information available in a short period of time can create cognitive difficulties for consumers, which will confuse consumers’ cognition . Second,Second, guidance shopping has a positive effect on cognitive assimilation but has no significant effect on attractiveness. Guidance shopping is an important factor in the theory of IT affordance. From the perspective of IT affordance, guidance shopping is to provide consumers with personalized product recommendations according to consumers’ needs and preferences for products. The two sides solve the problems generated by consumers in the shopping process through further exchanges, so as to better meet their needs. In the process of providing shopping guidance to consumers, the relationship between streamer and audience is more closely. From the perspective of products, the reason why consumers choose live streaming shopping is largely due to their own needs. Before making shopping choices, consumers often have a subjective judgment on whether the product is suitable for them. The director’s shopping guidance only makes consumers clear about their shopping choices, which explains the reason why guidance shopping has no obvious influence on attraction.Third, visibility has a significant positive impact on cognitive assimilation but has no significant impact on attraction. Visibility is one of the relevant technical characteristics in the field of social commerce. From the perspective of social commerce, visual perception is the most direct way for consumers to obtain goods or service information. Consumers produce direct impressions of the product through the information they see. The product information provided by visibility for consumers can effectively reduce consumers’ perceived risk and uncertainty of products . In the Fourth, media richness has a significantly positive impact on attractiveness and cognitive assimilation. In the live streaming situation, both businesses and consumers have rich media information expression. The streamer can introduce product information through text, pictures, and live streaming videos. The audience can also express their ideas through rich media . Media rFifth, attraction and cognitive assimilation have a positive impact on consumers’ purchase intention. Attraction is the highlight of live streaming shopping compared with other social business models. Due to its more entertaining nature, live streaming shopping meets consumers’ shopping needs and their own entertainment needs, which will bring more satisfaction to consumers. Meanwhile, the emotional connection based on interaction is often more solid than the single communication based on text, which can provide consumers with a sense of social existence. Online live streaming eliminates consumers’ uncertainty in the shopping process through more abundant product information displays. It also urges consumers’ cognition of products more comprehensive. This cognition is produced after consumers watch live streaming shopping. In terms of time, it is subjective cognition that can most affect consumers’ purchase intention. The survey results show that consumers’ own cognitive assimilation is an important prerequisite for influencing consumers’ behavior. Cognitive assimilation based on information acquisition will affect consumers’ subsequent social sharing and have an important impact on their willingness to consume.Finally, attraction and cognitive assimilation mediate the relationship between visibility, media richness, guidance shopping, real-time interaction, and consumer purchase intention. Attraction and cognitive assimilation can affect the impact of social and technical factors on consumer purchase intention. Technical factors and social factors in live streaming shopping are the direct factors affecting consumers’ purchase intention, which are realized by attraction and cognitive assimilation. Live streaming shopping mode attracts the attention of consumers with its own characteristics. Consumers form their own cognition through the acquisition of live streaming shopping information. This cognition is largely affected by technical factors and social factors in live streaming shopping mode. Attraction and cognitive assimilation change the influence of social and technical factors on consumers’ purchase intention to some extent.5.2.The theoretical contributions of this research mainly focus on the following aspects.First, from the perspective of socio-technical interaction, this study discusses the factors that affect consumers’ purchase intention in live streaming shopping. Although there have been studies from a technical point of view to explore the technical characteristics of live streaming shopping on the impact of consumer purchase intention, there has been a lack of research on the social characteristics of live streaming shopping . Most ofSecond, this study introduces consumer emotion into the study of consumer behavior and confirms that the emotional attitude of consumers in the process of live streaming shopping will greatly affect their final purchase decision. Although there have been studies on the factors affecting consumers’ behavior from many aspects, there is a lack of discussion on the influence of emotional tendencies on consumer decision-making . In the Third, this study expands the theoretical application of technology affordance theory in live streaming shopping situations. Although previous studies have researched the impact of IT affordance theory on consumer purchase behavior, with the support of information network technology, the new social commerce model has solved many problems that cannot be solved before . In the At the same time, based on the theory of IT affordance, this study also discusses the interaction between technical functions and social emotions in the live streaming scene and further refines the theoretical research on live streaming shopping.5.3.Through the discussion of the research results, the practical implications of this study mainly concentrate on the following aspects. First, this study helps enterprises to understand the relationship between social and technical characteristics in live streaming shopping scenes. For example, in terms of consumer perception, the richer product information is expressed, the more consumer uncertainty is eliminated . When diSecond, the research results help enterprises to better apply live streaming technology and expand the channels of product promotion. The great enrichment of material living standards has changed people’s purely demand-oriented shopping style. In social business activities, people pay more attention to the experience in specific scenarios. Enterprises involved in online shopping should shift their focus from “traffic” to “context” to meet the needs of consumers and create a more diversified live streaming shopping scene. As one of the marketing scenarios, live streaming can quickly attract consumers’ attention. How to create content product information around consumers’ psychology, fully connect with consumers’ values and create live streaming content according to consumers’ value needs is very important for product marketing of enterprises.5.4.The research of this study has the following limitations: First, the respondents in this study are people with the live streaming shopping experience and there is a lack of surveys on users who use other online shopping models. Whether the survey conclusion is applicable to consumers of other new online business models needs further demonstration. Second, due to the complexity of the live streaming shopping environment and the diversity of participants, consumers’ purchase intention cannot completely represent their final purchase behavior. Therefore, longitudinal studies on consumer behavior at different time periods can be added to future research, in order to have a clearer understanding of consumer behavior. Finally, streamers are also important factors affecting consumers’ purchasing behaviors in live streaming shopping. Their professional and trustworthy characteristics may affect consumers’ final purchasing decisions. Future studies can start with various subjects participating in live streaming shopping to study the influence of the characteristics of different subjects on consumers’ purchasing behaviors.6.From the perspective of socio-technical interaction, this study explores the technical and social factors that affect consumers’ purchase intention in live streaming shopping scenes. Through the study, we found that technical factors and social factors do not affect consumer purchase intention alone. Under the interaction of technical and social factors, the characteristic factors in live streaming shopping will affect consumers’ shopping choices. This study not only expands the theoretical research on consumer behavior in the context of live streaming shopping but also provides practical guidance for enterprises to better apply live streaming technology for product marketing.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.XD: conceptualization, theoretical foundation, and investigation. XL: methodology and writing. XX: writing. All authors contributed to the article and approved the submitted version.This work was supported by the grants from the National Natural Science Foundation of China 71902158, 72272122, 71972061, and 72172043. This work was supported in part by the Guangdong Basic and Applied Basic Research Foundation under grant 2019A1515110992.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.