ZnxCdx1−S/ZnS composites are composed of ZnS nanosheets decorated with ZnxCdx1−S nanorods, and TEM and UV-vis absorption spectra confirm the formation of the heterostructure between ZnxCdx1−S nanorods and ZnS nanosheets. Because of the well-matched band alignment, stronger optical absorption and larger carrier density, Zn0.2Cd0.8S/ZnS has the highest hydrogen production, with a photocatalytic hydrogen production rate up to 16.7 mmol g−1 h−1 under visible light irradiation. Moreover, the photocatalyst also exhibits high stability and good reusability for hydrogen production reaction. The facile and efficient approach for ZnS based heterostructures could be extended to other metal compound materials.Hydrogen evolution from water using solar energy is regarded as a most promising process, thus, exploring efficient photocatalysts for water splitting is highly desirable. To avoid the rapid recombination of photogenerated electrons and holes in CdZnS semiconductors, Zn 2 evolution mechanism for the Zn0.2Cd0.8S/ZnS nanocomposites.Schematic illustration for electron charge transfer and H Among various strategies for hydrogen evolution, photocatalytic hydrogen evolution is regarded as the most promising way from water splitting using solar energy, because of the direct utilization of solar energy to achieve H2 generation from water splitting, as well as its much simpler and more economically competitive systems.1In consideration of the global energy crisis and environmental issues, much effort has been made to replace exhaustible fossil fuels with clean energy sources. Hydrogen, with a fuel value  : S molar ratio of 1 : 3.5 has much higher photocatalytic activity and exhibited an excellent stability after six cycles. And the H2 production of (CdS–ZnS)–TiO2 supported photocatalytic system was studied by Tambwekar et al.8 The hydrogen evolution rate of ZnCdS–CdS heterostructure was obtained from 25.46 to 72.82 and 52.82 μmol h−1, and the value increased to 192.28 μmol h−1 after the adoption of VS2 to the heterostructure surface.9 GQDs/ZnCdS/PdS exhibited a H2 evolution rate of 517 μmol h−1, which is 15, 7 and 1.7 times higher than that of pure ZnCdS, GQDs/ZnCdS, and ZnCdS/PdS, respectively.10 α-Fe2O3/Zn0.4Cd0.6S heterostructure has visible light photocatalytic H2 production of 536.8 μmol h−1.11 The ZnCdS–CH solid solution without noble metal loading achieved a superior photocatalytic H2 activity rate of 0.971 mmol h−1 under visible light irradiation (λ ≥ 420 nm), which exceeds those of coprecipitated Zn0.5Cd0.5S samples by more than 13 times.12 The hydrogen production rate of Fex1−Ptx-ZnCdS NPs had a significant enhancement over the pure ZnCdS (740 μmol g−1 h−1). The hydrogen production activity of ZnCdS–NiCoP composite catalyst was improved greatly, which reached 5.2 times that of pure ZnCdS.13 The highest hydrogen production rate of 2.265 mmol g−1 h−1 was achieved by the 0.5 wt% Fe0.3Pt0.7–ZnCdS nanocomposites, which was even better than that of 0.5 wt% Pt–ZnCdS (1.626 mmol g−1 h−1) under the same condition.14Ternary ZnCdS nanomaterials were regarded as a promising substitute for noble-metal co-catalysts in photocatalytic reactions because of their tunable band gap energy and lattice constant between 0.331 (〈100〉 of ZnS) and 0.359 nm (<〈100〉 of CdS) in comparison with ZnS or CdS materials.2 evolution rate of Zn0.3Cd0.7S nanorods was improved from 517.4 to 3310.1 μmol g−1 h−1 by loading a suitable amount of Ni3C NPs as co-catalyst under visible light irradiation.15 Hollow Zn0.6Cd0.4S cage material exhibited the highest hydrogen production rate of 5.68 mmol h−1 g−1 under cocatalyst-free and visible-light irradiation conditions.16 The addition of NiB greatly improved the photocatalytic performance of CdZnS, and the hydrogen production of NiB/CdZnS catalysts reached 8.137 mmol g−1 h−1, which is 17 times that of pure CdZnS.17 A typical “type II” band alignment forms at Zn0.5Cd0.5S hybrided with carbon materials lead to the improved H2 generation rate of 10.8 mmol g−1 h−1, exceeding the Zn0.5Cd0.5S nanosheets (6.4 mmol g−1 h−1) and QDs (3.4 mmol g−1 h−1).18 A controlled ZnCdS QDs developed by a simple ZIF-8 templating method exhibited significantly enhanced photocatalysis performance with a H2 production rate of 3.70 mmol h−1 g−1 (Zn0.5Cd0.5S QDs),19 and the highest hydrogen production rate of Zn0.5Cd0.5S/dodecahedron ZIF-67 composite reached 23.264 mmol g−1 h−1 under visible light irradiation,20 which was explained as the matched valence band position for ZIF-67 and ZCS and the exposure of rich active sites. The WO3/ZnCdS compound catalysts showed the hydrogen production activity of the 35 wt% WO3/ZnCdS to 98.68 μmol mg−1, about 9.6 times that of pure ZnCdS (10.28 μmol mg−1).21The photocatalytic H2 production. In this paper, simple hydrothermal and annealing reaction methods were developed for preparation of pure ZnS nanosheets, CdS nanorods and ZnxCdx1−S/ZnS composites. The photocatalytic hydrogen production rate of Zn0.2Cd0.8S/ZnS (#3) up to 16.7 mmol g−1 h−1 has been obtained under visible light irradiation with a good stability. This value is larger than the reported pure ZnxCdx1−S/ZnS composites. Furthermore, the preparation method is simple. The outstanding photocatalytic performance benefited from the efficient charge transfer between ZnCdS nanorods and ZnS nanosheets in the ZnxCdx1−S/ZnS heterojunctions, as well as more active sites on the surface of nanosheets. This study will be helpful to obtain catalysts for photocatalytic H2 production based on low-priced metal composite materials.Although many efforts have been done to develop efficient photocatalytic systems and photocatalysts by using ZnCdS materials and their composites, it is still an urgent task to develop low-cost co-catalysts for advancing photocatalytic H2·2H2O], cadmium acetate [Cd(OAc)2·2H2O], thiourea [(NH2)2CS] and ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai China). Ethylenediamine was purchased from Aladdin Chemical Reagent Co., Ltd (Shanghai).All chemicals were analytical-grade and used without further purification. Zinc acetate  direction. At the same time, the lattice spacing of 3.13 Å in the nanosheet matched well with the (002) plane of hexagonal ZnS. The result is in good agreement with XRD. From elemental mappings in xCdx1−S nanorods, and ZnxCdx1−S nanorods were sparsely distributed on the surface of ZnS nanosheets.TEM and HRTEM measurements were carried out to investigate the microstructure features, and HAADF-STEM to determine the elements distribution. TEM images of selected ZnxCdx1−S heterostructures with ZnS nanosheets decorated with ZnxCdx1−S nanorods.Therefore, from the XRD patterns, SEM and TEM images show that samples #2 and #3 were composed with ZnS/Zn26,27 A decrease in the fluorescence intensity in samples #2 and #3 implies the suppressed recombination of photogenic electron–hole pairs upon attaching ZnxCdx1−S nanorods on ZnS nanosheets, and an increased photogenerated electron–hole separation, and the shift of peak position probably attributed to the transition at the interface.The PL spectra were carried out to study the properties of electrons' recombination light emission. As displayed in xCdx1−S. The observed two absorption edges in samples #2 and #3 reveal the formation of the ZnxCdx1−S/ZnS heterostructures. Sample #3 has a stronger absorption and bigger absorption edge than sample #2. Then, the band gap energy is obtained as 3.46, 2.48, 2.35 and 2.19 eV for samples #1–#4, respectively.UV-vis-NIR absorption spectra  reveal t2 g−1 for pure ZnS nanosheets (#1), and 103.2, 82.3 and 20.9 m2 g−1 for #2, #3 and #4, respectively. From the results, one can find that pure CdS nanorods possess a low BET surface area, and sample #3 has larger BET surface than sample #2, which is favorable to adsorb molecules.The hollow porous structures of the as-prepared samples were elucidated by nitrogen adsorption/desorption measurements to obtain their BET surface areas. As shown in 3/2 and 2p1/2 of Zn2+, as shown in xCdx1-S/ZnS heterostructures  has a slightly shift to higher energy, indicating the decreased electron density around Zn atoms.28 The Zn 2p3/2 core level spectra can be deconvolved into two peaks, as shown in 2+ in the sulfur deficient regions.29 The relative intensity ratios between the higher one and total zinc are equal to 0.298, 0.280 and 0.285 for sample #1, #2 and #3, respectively. That means, many sulfur vacancies exist in sample #2 and #3, and the concentration is approximate, consistent with the EDX results. The Cd 3d XPS spectra are shown in 5/2 and 3d3/2, respectively, and there is no obvious peak shift between pure CdS nanorods and ZnxCdx1-vS/ZnS heterostructures. For S 2p, the peaks at about 161.0 eV and 162.2 eV are belong to S 2p3/2 and S 2p1/2, as shown in xCdx1−S. The above XPS analyses demonstrated that the electron transformation from Zn and Cd to S in ZnxCdx1−S/ZnS heterostructures can increase electron density of active sites and beneficial to the various reactivity.30,31XPS measurements were used to investigate the surface composition of the four samples, the binding energies have been calibrated using the carbon C 1s peak (285.0 eV) as reference. Zn 2p spectra exhibited two peaks at ∼1020.9 eV and 1043.8 eV, which were assigned to Zn 2pThe apparent quantum efficiency was measured using the 300 W xenon lamp with a 420 nm band pass filters and calculated according to 2 production was carried out under visible light irradiation (with a 420 nm cut filter) for samples ZnS, CdS and ZnxCdx1−S/ZnS, as shown in xCdx1−S nanorods reveal an enhanced photocatalytic activity. Sample Zn0.3Cd0.7S/ZnS has a hydrogen production rate of 9.2 mmol g−1 h−1, while Zn0.2Cd0.8S/ZnS (#3) achieved a H2 production rate up to 16.7 mmol g−1 h−1 with an apparent quantum efficiency of 10% at 420 nm, approximately 1.5 times of the former. The value is nearly three times of pure CdS nanorods (5.9 mmol h−1 g−1).32 This result is larger than recently reported 10.8 mmol g−1 h−1 with Zn0.5Cd0.5S hybrided with carbon materials as catalysts,18 but smaller than the value of 23.264 mmol g−1 h−1 with Zn0.5Cd0.5S/dodecahedron ZIF-67 composite.20 Moreover, different from the reported severe photocorrosion happened in CdS materials,33–35 the as-prepared Zn0.2Cd0.8S/ZnS heterostructures have a relatively stable H2 reproduction with seven consecutive cycles  for the four sample, as depicted in 37e0, ε and ε0 are the electron charge with a value of 1.6 × 1019 C, dielectric constant with a value of 10,38 and permittivity of vacuum with a value of 8.85 × 10−12 F m−1, respectively. A, V and Cs are the area of the film electrode, the bias applied on the electrode and surface capacitance, respectively. From 0.2Cd0.8S/ZnS (#3) has the smallest value of slope, so it has the highest carrier concentration up to 2.42 × 1019, larger than the value of 1.95 × 1019, 1.98 × 1019 and 1.88 × 1019 cm−3 for sample #1, #2 and #4, respectively. The higher carrier concentration results from the sulfur vacancy. Therefore, the enhanced H2 production rate of sample #3 can be ascribed to the improved charge transfer activity. Besides, the flat-band potentials derived from the M–S plots are equal to −0.474, −0.433, −0.558 and −0.568 V versus Ag/AgCl electrode at neutral solution for #1, #2, #3 and #4, respectively, that correspond to the related conduction band versus Ag/AgCl electrode.39Mott–Schottky (M–S) plots were explored to determine the flatband potential , electrons will be excited from the conduction band of ZnCdS nanorods to the valence band due to the lower band gap energy. As the conduction potential of ZnCdS is more negative than that of ZnS, the excited electrons will transit from the conduction band of ZnCdS nanorods to that of ZnS nanosheets, resulting in the separation of electron–hole pair. Due to the existence of internal fields, the potential barrier at the interface of the heterostructure prevents photogenerated back to ZnCdS nanorods, the photogenerated electrons have a longer lifetime. At the same time, sample #3 has higher electron carriers, meaning that the electrons could be rapidly transmitted to start reduction reaction with H2O, producing hydrogen, while the hole sacrificial agent l-lactic acid in the system consumed electrons, forming pyruvic acid and hydrogen (h+ + l-lactic → CH3COCOOH + H2). These phenomena effectively promoted the photoinduced electron–hole separation and resulted in the excellent photocatalytic performance. The better photocatalytic H2 reproduction of sample #3 than sample #2 can be ascribed to the lower charge transfer resistance and the stronger optical absorption, the lower charge transfer resistance will accelerate electrons' transition to participate the reduction reaction with H2O; and the stronger visible light absorption of sample #3 can provide more photogenerated electrons for hydrogen production (2H+ + 2e− → H2).In consideration of band gap energy and the conduction band potential xCdx1−S/ZnS composites were synthesized via simple hydrothermal and annealing reaction methods. ZnxCdx1−S/ZnS heterostructures were composed with ZnS nanosheets decorated with some ZnxCdx1−S nanorods. Type-II Zn0.2Cd0.8S/ZnS heterostructures have a lower charge transfer resistance, and stronger optical absorption, as well as a well-matched band energy alignment, hence they achieved a H2 production rate up to 16.7 mmol g−1 h−1 under visible light irradiation (λ > 420 nm), approximately 1.5 and 3 times higher than that of Zn0.3Cd0.7S/ZnS (#2) and pure CdS nanorods (5.9 mmol h−1 g−1) respectively. The strategy for the construction of heterojunctions can provide more perspective for the preparation of photocatalyst in different application field with a simple preparation method.In summary, pure ZnS nanosheets, CdS nanorods and ZnThe authors declare no competing financial interest.


The framework stability and thermal robustness of prepared materials were investigated using thermogravimetric analysis (TGA) and high-energy powder X-ray diffraction (HE-PXRD). MOFs were tested as adsorbents for different gases at various pressures and temperatures. Nitrogen and argon adsorption showed that the activated samples have moderate BET surface areas: 103 m2 g−1 (N2)/126 m2 g−1 (Ar) for UPJS-7′′, 320 m2 g−1 (N2)/358 m2 g−1 (Ar) for UPJS-9′′ and UPJS-8′′ adsorbs only a limited amount of N2 and Ar. It should be noted that all prepared compounds adsorb carbon dioxide with storage capacities ranging from 3.9 to 2.4 wt% at 20 °C and 1 atm, and 16.4–13.5 wt% at 30 °C and 20 bar. Methane adsorption isotherms show no adsorption at low pressures and with increasing pressure the storage capacity increases to 4.0–2.9 wt% of CH4 at 30 °C and 20 bar. Compounds displayed the highest hydrogen uptake of 3.7–1.8 wt% at −196 °C and 800 Torr among MTB containing MOFs.A series of four novel microporous alkaline earth metal–organic frameworks (AE-MOFs) containing methanetetrabenzoate linker (MTB) with composition {[Ca Four novel microporous alkaline earth metal–organic frameworks (AE-MOFs) containing methanetetrabenzoate linker (MTB): UPJS-6, UPJS-7, UPJS-8 and UPJS-9 have been successfully prepared, characterized and tested as adsorbents for different gases. Hydrogen storage in MOF is currently being studied intensively. Hydrogen as a technologically important gas provides an ideal clean energy source, since no detrimental molecules are produced causing an environmental burden. Hydrogen is the smallest and simplest gas molecule with a very low boiling point, and its storage and transportation present a challenge.6 The second technologically important gas is carbon dioxide, which is industrially used to prepare many intermediate or fine chemicals. CO2 is also the main component of greenhouse gases and represents one of the largest anthropogenic impacts on the environment. For this reason, it is necessary to reduce its concentration in the atmosphere and thus limit global warming.7 Physical absorption of hydrogen and carbon dioxide in porous MOFs represents an interesting approach to the storage of the mentioned gases.Metal–organic frameworks (MOFs) represent a class of inorganic–organic hybrid materials constructed from metal ions or clusters and organic linkers to form porous polymeric frameworks with interesting properties. Due to their structural and functional diversity, large surface area, porosity and affinity toward various guest molecules, MOFs have been tested as materials for different applications including gas storage and separation,8 Carboxylate linkers with tetrahedral symmetry are highly suitable for MOF preparation, because they form the desired porous frameworks with large surface areas. To date, many MOFs have been prepared that contain ligands that possess tetrahedral geometry: methanetetrabenzoate (MTB),9 methanetetrabiphenylcarboxylate,10 methanetetrakistetraacrylate,11 methane tetrakistetrakistetrabenzoate11 and tetrakis{3,5-bis[(4-carboxylate)phenyl]phenyl}methane.12 The chemical and physical properties of AE-MOFs have found applications in gas storage and separation,13 catalysis,14 sensing15 and proton conduction.16 Numerous AE-MOFs have been explored predominantly for gas storage of carbon dioxide, hydrogen and also for nitric oxide, noble gases and hydrocarbons.13Alkaline earth metal–organic frameworks (AE-MOFs) are investigated relatively less compared to MOFs that contain transition metals, although they display several unique advantages such as: (a) high abundance in earth's crust, (b) low cost, (c) low toxicity , (d) low density  that offers gravimetric benefit for storage applications, (e) stability in air and (f) ionic nature leading to strong interactions with negative functional groups such as carboxylate. Unlike the transition metals, which tend to form largely predictable secondary building units (SBUs), the bonds between alkaline earth metal centres with functional groups are predominantly ionic in nature and the prediction and control over coordination geometry is ambiguous. The nature of organic linker plays an important role in the overall dimensionality and topology of the frameworks. Carboxylate-based linkers are those used most commonly to synthesize AE-MOFs due to the strong interaction between carboxylate oxygen atoms and alkaline earth metals. Based on the HSAB theory, AE metals fall under the hard acids and coordinate well with carboxylate functionalities.ii),17,18 Ni(ii)19 and Pb(ii)20,21 metal centres and tested them as adsorbents for gases and heterogeneous catalysts. In the course of our continuing investigation into the crystal engineering of MOFs containing MTB linker and their applications, we combined the advantages of AE metals with MTB. Four novel MOFs were successfully prepared, characterized and tested as adsorbents for nitrogen, argon, carbon dioxide, methane and hydrogen at different temperatures and pressures.In our previous studies, we reported several compounds containing tetrahedral methanetetrabenzoate linker (MTB) with Zn2·4H2O) for synthesis of compounds UPJS-6 and UPJS-7, 43 mg 2) for compound UPJS-8 and 53 mg 2) for synthesis of compound UPJS-9 were used. H4MTB was dissolved in 3 cm3 of N,N′-dimethylformamide (DMF), followed by the addition of the appropriate metal nitrates and then 3 cm3 of ethanol and 2.8 cm3 of water were added while stirring the solutions until the reactants were completely dissolved. Prepared solutions were sealed in glass autoclaves and the temperature programme was controlled by a thermoregulator. The temperature mode was set as follows: the reaction mixtures of compounds UPJS-6, UPJS-8 and UPJS-9 were heated to 80 °C and UPJS-7 to 110 °C with a heating rate of 0.5 °C min−1 for 72 hours. After this time, the reaction vessels were cooled to room temperature with a cooling rate of 0.5 °C min−1. Prepared materials were filtered off, several times washed with mixture of solvents used in the synthesis and dried in the stream of air, yields: 17.8 mg of UPJS-6 (yield 38%), 17.5 mg of UPJS-7 (yield 28%), 18.5 mg of UPJS-8 (yield 35%) and 22.5 mg of UPJS-9 (yield 35%). Elemental analysis for UPJS-6  clcd: C, 56.38%; H, 3.99%; N, 2.05%; found: C, 57.14%; H, 4.05%; N 1.98%; for UPJS-7  clcd: C, 49.32%; H, 5.07%; N, 4.15%; found: C, 48.43%; H, 5.10%; N 4.23%; for UPJS-8  clcd: C, 46.94%; H, 4.68%; N 3.95%; found: C, 46.51%; H, 4.72%; N, 3.96% and for UPJS-9  clcd: C, 40.25%; H, 3.96%; N 1.90%; found: C, 40.86%; H, 3.87%; N, 2.01%. Assignment of characteristic absorption bands (in cm−1) in IR spectra for UPJS-6: ν(OH) 3409; ν(CH)ar. 3069, 3038; ν(CH)aliph. 2963, 2932, 2891; ν scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O) 1664; ν(CC) 1600; νas(COO−) 1543; νs(COO−) 1407; UPJS-7: ν(OH) 3423; ν(CH)ar. 3068, 3040; ν(CH)aliph. 2996, 2933, 2890; ν(CO) 1670; ν(CC) 1660; νas(COO−) 1547; νs(COO−) 1408; UPJS-8: ν(OH) 3412; ν(CH)ar. 3067, 3039; ν(CH)aliph. 2959, 2931, 2887; ν(CO) 1662; ν(CC) 1598; νas(COO−) 1541; νs(COO−) 1398; UPJS-9: ν(OH) 3407; ν(CH)ar. 3064, 3037; ν(CH)aliph. 2957, 2929, 2884; ν(CO) 1660; ν(CC) 1590; νas(COO−) 1537; νs(COO−) 1392 cm−1. Corresponding IR spectra of prepared compounds are depicted in Fig. S1 in ESI.‡The compounds were prepared by solvothermal synthesis in glass Ace® autoclaves. In the synthesis, the nitrates of the respective cations and methanetetrabenzoic acid were used in a molar ratio 5 : 1.20 mg (0.04 mmol) of methanetetrabenzoic acid and 48 mg  for easier activation of compounds, the solvent exchange process was performed. 100 mg of as-synthesized samples were gently grinding and then dispersed in 30 cm2.2The elemental analysis was performed with a CHNOS Elemental Analyzer vario MICRO from Elementar Analysensysteme GmbH with ±2–3 mg of sample.−1. The samples were prepared in the form of a KBr pellet with a complex/KBr mass ratio of 1/100. Before IR measurements KBr was dried at 700 °C for 3 h in an oven and cooled in a desiccator.The infrared spectra were recorded with Avatar FT-IR 6700 spectrometer in the range 4000–400 cm2O3 crucible and heated under a dynamic atmosphere of air (60 cm3 min−1) with a heating rate of 10 °C min−1, using the TGA Q500 apparatus.Thermal behaviour of as-synthesized and solvent exchanged samples was studied by thermogravimetry analysis (TGA) in the temperature range of 30–800 °C. The sample with a weight of approximately 20 mg was placed in Alin situ heating with a heating rate of 10 °C min−1 in an atmosphere of argon and beam energy of 60 keV, corresponding to a wavelength of 0.207150 Å. The diffracted photons were collected using an image plate detector PerkinElmer  carefully aligned orthogonal to the X-ray beam. The sample-to-detector distance and the detector tilt were determined from diffraction patterns obtained from CeO2 standard reference material. The two-dimensional diffraction patterns were integrated using the QXRD software. The observed X-ray scattering intensities were corrected for background, absorption, polarization, incoherent and multiple scattering effects.HE-PXRD measurements were carried out at the P01 wiggler beamline of the PETRA III positron storage ring in DESY . The patterns were collected during 2.319786381978639197863719786362 using the Shelx program suite.22 Nonhydrogen atoms were refined anisotropically. Hydrogen atoms were included in idealized positions and refined isotropically. The contribution of guest molecules located in the pores was subtracted by the SQUEEZE procedure in Platon.22 The figures of final crystal structures were drawn using the DIAMOND software. Detailed crystallographic data are given in Table S1 in ESI‡Single-crystal diffraction data for UPJS-6  basis set for all atoms.2.5p/p0 = 0.005 to 0.95. Based on the nitrogen adsorption isotherm, the BET specific surface area (SBET) and pore volume (Vp) of the samples were calculated.Nitrogen adsorption isotherms measured at −196 °C were taken using Quantachrome Nova 1200e automatic adsorption analyser. Before the adsorption studies, the solvents located in the channel system of samples were exchanged by ethanol (for procedure see Section 2.1.2 Solvent exchange process) and further, the samples were activated by the evacuation at 100 °C for 24 h to remove the guest molecules. Activated samples were denoted as UPJS-7′′, UPJS-8′′ and UPJS-9′′. The nitrogen adsorption isotherms for desolvated samples were collected in a relative pressure range from Argon adsorption isotherms were measured at −186 °C, hydrogen adsorption isotherms at −196 °C and carbon dioxide adsorption isotherms were performed at 20 °C using ASAP 2020 apparatus.2 and CH4 using a homemade high-throughput instrument.24 Gas adsorption experiments were measured via a manometric gas dosing system on three samples in parallel. The amounts of gas adsorbed were calculated by an equation of state using the Reference Fluid Thermodynamic and Transport Properties (REFPROP) software package 8.0 of the National Institute of Standards and Technology (NIST).25 The high pressure adsorption isotherms were measured on the same batch of the sample as nitrogen adsorption. Samples were thermally activated individually in situ under the primary vacuum, at 80 °C overnight.The gas adsorption measurements at 30 °C and pressures up to 20 bar were performed with CO2 and CH4) two measurements with the same parameters were performed on the same sample. Between the first and the second experiment, the samples were submitted to an evacuation step at 30 °C and under the primary vacuum during 80 minutes. In this way, from the second gas adsorption measurement, the regenerability/recovery of the samples was checked under the mentioned conditions.The regenerability of the samples has been evaluated under mild conditions. For each gas  compounds UPJS-6 and UPJS-7, the influence of the reaction temperature was observed. In the synthesis of these materials, the same reaction conditions were used except temperature. Compound UPJS-6 was prepared at 80 °C and represents the low-temperature phase and compound UPJS-7 was synthesized at 110 °C, which can be referred as the high temperature form. The temperature difference of 30 °C had a significant effect on the mutual arrangement of the building components, which led to the formation of different polymeric frameworks with different porosity .All the compounds investigated in the present study were prepared using a solvothermal synthesis under mild synthetic conditions (80–110 °C) and in a molar ratio of 5 : 1. It is known that the synthesis of MOF may be included in so-called combinatorial synthetic chemistry since the successful preparation of porous coordination polymers depends on many factors such as: reactant concentration, the presence of a co-solvent, solution pH, metal to ligand ratio, properties of the used counterion, reaction temperature, and reaction time, can have a considerable impact on the final composition of the products.The exact formula and composition of all prepared samples were established based on the combination of single-crystal X-ray diffraction experiments, elemental analysis and thermogravimetric analysis results.3.23.2.14] donor set is coordinated by four oxygen atoms O1, O4, O9 and O11 from the four different coordinated carboxylate groups of MTB4− ligands. The oxygen atoms O1 and O9 originated from two COO− groups with the carbonyl atoms C1 and C30 which are coordinated in the chelate-anti–anti mode and two oxygen atoms O1 and O3 from carboxylates C13 and C42 are coordinated in syn–syn mode. The resulting polyhedron geometry of Ca3 could be described as a deformed planar square (see 7] donor set is heptacoordinated by oxygen atoms of four carboxylate moieties coordinated in syn–syn (carbonyl atom C56), chelate-anti  and chelate-anti–anti (C30) fashion and the coordination polyhedron around Ca4 could be described as a deformed hexagonal pyramid. Since the hexagonal basal plane is made up of three chelate-coordinated carboxylate ligands whose bite angle is limited, the angles of O–Ca–O differ from an ideal value of 60° for a hexagon. As an example, the chelate angle O5–Ca4–O6 having the highest value of 52.65(12)°  and one carboxylate group in syn–syn (C27) mode. Oxygen O15 of the second syn–syn (C56) carboxylate group completes the pyramidal top of the polyhedron. Carboxylate groups with carbonyl atoms C49 and C27 form a bridge between the calcium ions Ca1 and Ca2. The central atom Ca2, in contrast to the other crystallographically independent calcium ions, is six coordinated with deformed pentagonal pyramidal geometry 7(μ3-COO)] are arranged in the Ca3–Ca4–Ca1–Ca2 sequence with Ca⋯Ca distances: 3.8746(16) Å (Ca3⋯Ca4), 3.7481(16) Å (Ca4⋯Ca1) and 4.1509(17) Å (Ca1⋯Ca2) . Analysis of the framework using the ToposPro program showed that the compound did not imitate any known mineral and the skeleton topology could be designated as 3,4^2,6,8-c net.27 Since compound UPJS-6 contains isolated pores in the crystal structure, its gas adsorption properties were not studied.The SC-XRD analysis showed that in the crystal structure of compound UPJS-6 isolated cages are present. Although the inner diameter of the cage is relatively large with a size of 18.3 × 8.4 Åsize see . The tot3.2.24(μ4-O)(μ2-COO)8(H2O)4], in which four Ca ions share a common O1 oxygen atom (μ4-binding), which is located in a special position on a four-fold axis. It is of interest for this cluster that the bridge oxygen atom is not tetrahedrally surrounded, as in various modifications of the MOF-5 compounds (IRMOFs28), basic beryl or zinc acetate. The axially coordinated oxygen atoms are located in the corners of the square planar cluster with O–Ca–O angle of 90° and the distance between central atoms Ca⋯Ca is 3.797(4) Å (see Table S3 in ESI4(μ4-O)(μ2-COO)8(H2O)4] cluster are coordinated in chelate-anti coordination mode and the secondary building unit have a cubic shape  −x, y, 2 − z, anti-coordination) is longer, 2.626(4) Å (see Table S3 in ESI4-O coordinated oxygen coordinated to Ca atom (2.685(3) Å (Ca–O1)). The coordination sphere of each central atom is completed by a coordinated water molecule with a Ca–O4 bond length of 2.274(9) Å. The oxygen atoms of the coordinated aqua and oxido ligands form the pyramidal caps of the trigonal prism. In the framework of UPJS-7 with two types of openings are present having a size of 12.6 × 9.4 Å2 and 9.4 × 8.0 Å2 propagating along a and b crystallographic axis  structure. The central carbon atom of the MTB ligand can be considered the F vertex and the [Ca4(μ4-O)(μ2-COO)8(H2O)4] cluster decorates the calcium(ii) positions in the CaF2 structure.The topological analysis see  of the p3 (cell volume 4689.0 Å3) and corresponds to 57.7%.The total free space in the crystal structure of UPJS-7, after removal of the guest molecules and analysed by PLATON, is 2529 Å3.2.3ii) ions are present which form the isolated triangular cluster with composition [Sr3(μ3-COO)2(μ2-COO)2(COO)2] (see ii) ions. Thus, oxygen atoms O1 and O2 provide all possible electron pairs to form coordination bonds. The bond distances of μ3-coordinated oxygen atoms and strontium ions are in the range of 2.573(3) Å to 2.802(3) Å  ligands.30In the crystal structure of compound UPJS-8, three crystallographically independent strontium(O)2] see . The dis7] donor set is coordinated by the seven oxygen atoms derived from the four different carboxylate moieties of the MTB4− ligand and the shape of the coordination polyhedron can be described as a deformed monocapped octahedron. Strontium ion Sr2 is also heptacoordinated by the seven oxygen atoms, but the shape of the coordination polyhedron [SrO7] is a deformed monocaped trigonal prism. The last strontium ion Sr3 is hexacoordinated with [SrO6] donor set and the shape of the coordination polyhedron is deformed octahedron 3/2]}n is formed. The polymeric framework contains 2D cavities with sizes of 15.5 × 5.0 Å2 propagating along a crystallographic axis and smaller openings with dimensions of 5.8 and 3.5 Å2 propagating along b axis (see 3 free volume of 9398.2 Å3 unit cell volume). Using the ToposPro program,27 an analysis of the polymeric framework was performed, which showed that the compound does not imitate any known mineral and is unique. The analysis showed that the framework has a 4,5^2,6,8-c net topology.By described coordination of strontium ions and MTB molecules, 3D coordination polymer with the composition of {[Sraxis see . The tot3.2.4ii) ions form a linear cluster with the composition [Ba3(μ2-COO)6(H2O)6]. The cluster is composed of two crystallographically independent barium ions Ba1 and Ba2 which are in the structure of compound UPJS-9 arranged in the Ba2–Ba1–Ba2i sequence (where (i) −x, y, 1/2 − z). Ba2–Ba1 are bridged together by three carboxylate ligands: two coordinated in chelate-anti mode  and one carboxylate group in syn–syn mode (C13). The central atoms are located in a special position and lying on a c slide plane (see ii) ions in the cluster [Ba3(μ2-COO)6(H2O)6] is 4.4208(4) Å. The described coordination of the carboxylate ligands on the central atoms forms a secondary building unit (SBU) with a connectivity of six and deformed tetragonal bipyramid shape. The central Ba1 atom is hexacoordinated by oxygen atoms with [BaO6] donor set and the shape of a polyhedron could be described as deformed tetragonal bipyramid  = −x, y, 1/2 − z) originated from coordinated carboxylate groups in chelate-anti mode and two oxygen atoms O2 and O2i from two carboxylate groups coordinated in syn–syn fashion. The carboxylate functional groups  also form a bridge between Ba1 and Ba2. The coordination sphere of the central atom Ba2 is formed of eight oxygen atoms: three oxygen atoms of coordinated terminal aqua ligands  with bond length range from 2.807(3) to 2.849(5) Å, oxygen atoms O1, O2, O5, and O6 derived from the carboxylate groups coordinated in the chelate-anti mode and oxygen O3 from carboxylate coordinated in syn–syn fashion. The bond lengths of the carboxylate oxygen atoms and Ba2 are comparable to those of coordinated aqua ligands  ions as building blocks results in a three-dimensional framework with 1D channels propagating along the c crystallographic axis  structure similar to UPJS-7. The [Ba3(μ2-COO)6(H2O)6] cluster decorates the calcium(ii) positions and the central methane carbon atom of the MTB ligand can be considered the fluoro vertex in fluorite structure.The combination of MTB molecules and Ba.Using PLATON analysis the total free pore volume of 2973.5 Å3.32O molecules). Similar high thermal stability and release of solvent molecules was observed not only for MOF compounds13 but also for common chemicals 2·H2O = 180–200 °C). This phenomenon can be explained by the strong interaction between water molecules and the central atoms. We can only speculate that there is such a strong interaction in UPJS-6, but the main reason for the higher thermal stability (210 °C) is the presence of isolated cages/pores in the framework. The water molecules located in them are trapped and are released only after the collapse of the framework. Mentioned explanation is also in good agreement with the results of the HE-PXRD analysis (see below), where the compound is stable at 200 °C and the polymeric skeleton collapses at 250 °C, allowing water molecules to be released, which was reflected by weight loss on the TG curve. TG curve of UPJS-7 showed a solvent weight loss of 32.49% in the 30–400 °C temperature range (expected 32.29% for 4 DMF and 8H2O molecules) and TG curve of UPJS-8 gave a similar solvent weight loss of 29.85% in the 30–300 °C temperature range (expected 29.47% for 4 DMF and 7H2O molecules). For compound UPJS-9 TG analysis gave a distinct weight loss of 22.17% in 30–300 °C temperature range corresponding to the loss of 10 water and 2 DMF molecules (expected 22.09%). After the desolvation process, frameworks exhibit high thermal stability up to 500 °C for UPJS-7 and UPJS-9 and 530 °C for UPJS-8. The high thermal stability of polymeric skeletons can be explained by the strong interactions formed between carboxylate oxygen atoms of MTB linker and AE central ions. The TG curves of ethanol exchanged samples UPJS-7′, UPJS-8′ and UPJS-9′ are shown in Fig. S2b in ESI.The thermal behaviour and stability of prepared compounds were investigated using thermogravimetry (TG) and obtained TG curves are shown in Fig. S2a in ESI.in situ heating and 50 °C steps. Fig. S3a–S6a in ESI31UPJS-6 can be included in the 1st MOF generation, which is sustained only with guest molecules and shows irreversible framework collapse on the removal of guest molecules. The polymeric skeleton of UPJS-7  in UPJS-7 and UPJS-8 occur in the temperature range 30–300 °C (based on TG) and frameworks stability is up to 450 °C (UPJS-7) and 500 °C (UPJS-8) without phase change (based on HE-PXRD), described compounds could be included into the 2nd MOF generation with stable and permanent polymeric frameworks classified by Kitagawa. Compound UPJS-9 exhibits similar thermal stability compared to previous samples. Moreover, a phase transition was observed  experiments with 3.4To characterize the porosity of prepared MOFs, gas adsorption measurements were performed for nitrogen, argon, hydrogen, methane and carbon dioxide. Before adsorption experiments, the samples were immersed in ethanol for one week to exchange the original solvents in the cavities (DMF and water), for easier activation of the compounds. The samples for gas adsorption experiments were prepared by evacuation at 100 °C for 24 h to remove all guest solvent molecules and activated samples were denoted as UPJS-7′′, UPJS-8′′ and UPJS-9′′.2 g−1; pore volume 0.048 cm3 g−1 (based on Ar) and 103 m2 g−1; pore volume 0.045 cm3 g−1 (based on N2) for UPJS-7′′, 358 m2 g−1; pore volume 0.256 cm3 g−1 (based on Ar) and 320 m2 g−1; pore volume 0.243 cm3 g−1 (based on N2) for UPJS-9′′. Compound UPJS-8′′ adsorbs only a limited amount of N2 and Ar and displayed no porosity. The surface areas of the compounds containing MTB linker are summarized in ii)33 and cobalt(ii)34 analogues.Textural properties of materials were initially studied by argon see  and nitr2 and framework.37 Compounds adsorb CO2 with maximal storage capacity of 3.92 wt%  for UPJS-7′′, 2.64 wt%  for UPJS-8′′ and 2.41 wt%  for UPJS-9′′ at 20 °C and 1 atm (760 Torr). The storage capacity of prepared compounds decreases in order UPJS-7′′ > UPJS-8′′ > UPJS-9′′. Calcium MOF (UPJS-7′′) adsorbs the highest amount of CO2 from prepared compounds, compared to UPJS-9′′ with the largest surface area (358 m2 g−1). As was mentioned in the introduction, AE-MOFs made of early members of the metal group are thought to be good candidates based on their gravimetric advantages over commonplace first-row transition metal-based MOFs. Surprisingly, the strontium complex UPJS-8′′ which does not adsorb nitrogen or argon, it adsorbs carbon dioxide. Thus, this compound exhibits a high selectivity for CO2 over N2 or Ar and can find its further application as a gas separator. High CO2/N2 selectivity was also observed for Zn-MOF38 and UPJS-1.39 A likely explanation is that nitrogen molecules cannot enter the pores at −196 °C due to large diffusional resistances, but at 20 °C the additional thermal energy allows the CO2 molecules to overcome these resistances. Moreover, higher quadrupole moment of CO2 might induce stronger interaction with the framework of UPJS-8′′.The carbon dioxide adsorption isotherms of prepared materials measured at 20 °C show type I behaviour with a hysteresis loop see . We supp2 adsorption capacity with reported compounds containing MTB ligand is summarized in 2 for presented AE-MOF appear to be low, but is not suitable to correlate them with other MTB-containing materials because their CO2 adsorption isotherms were measured at 0 °C. A comparison of carbon dioxide capacities can be performed with other MOF compounds that contain alkaline earth metals and measurements were performed under the same conditions, 1 atm and 20 °C. The observed value for UPJS-7′′ is comparable to Ca(SBD) ,40 Ca2(TIA)2(H2O)2 (4.90 wt%)41 but lower than that of compounds Ca5(BTB)2(HBTB)2(H2O)6 ,42 Mg-MOF-74 7 and Mg-DOBDC .7 Samples UPJS-8′′ and UPJS-9′′ show lower CO2 adsorption capacity values compared to the already published compounds containing strontium or barium ions such as Sr(SBA) 43 and Ba(HBTB) .44A comparison of the CO−1, 108.7 cm3 g−1) for UPJS-7′′, 2.17 wt%  for UPJS-8′′ and 1.80 wt%  for UPJS-9′′ at −196 °C and 800 Torr. Observed values of hydrogen storage capacities are relatively high and can be explained by the well-known high affinity of hydrogen to AE metal ions.13 Another explanation for the increased affinity of hydrogen for the presented MOFs is the presence of coordinatively unsaturated sites (CUSs) in UPJS-7 and UPJS-9. It is known that MOFs with CUSs offer interesting possibilities for tuning the affinity of these materials towards certain adsorbates, potentially increasing their selectivity and storage capacity. In MOFs with CUSs, at least one available coordination site of the metal centre is occupied by an atom belonging to a solvent molecule after the synthesis. Through activation process using evacuation, or heating, the solvent molecules can be removed, leaving the metal atom with open coordination site. UPJS-7 contains four terminally coordinated water molecules per one cyclotetranuclear cluster [Ca4(μ4-O)(μ2-COO)8(H2O)4], and UPJS-9 contains six water molecules in [Ba3(μ2-COO)6(H2O)6] cluster . Upon activation of the compounds, these solvent molecules are removed, leading to the formation of CUSs. Formed active sites serve as primary adsorption sites for hydrogen where an interaction central atom-hydrogen is present, thereby increasing the adsorption capacity of hydrogen in MOFs. Although UPJS-9 contains a higher number of CUSs compared to UPJS-7, its hydrogen sorption capacity is lower . This observation can be explained by the higher relative atomic mass of barium compared to calcium, which is reflected in the resulting values of the volume of adsorbed hydrogen, since in the case of adsorption measurements the amount of gas is given in units of cm3 per one gram of support. In compound UPJS-8, no terminally coordinated solvent molecules are present. However, the DFT modeling23 has shown that there is one unoccupied orbital on each central atom, which can serve as CUSs (κ4-L1)2]·2DMF·7H2O}n (UPJS-4)18 and {[Ni2(μ4-MTB)(κ4-L1)2]·4DMF·8H2O}n32 . Their hydrogen adsorption capacities at −196 °C were 1.28 and 0.70 wt%, respectively. It indicates that all novel prepared compounds exhibit significantly larger hydrogen uptake.According to the literature data, the H2 and CH4 isotherms measured at 30 °C, pressure up to 20 bar and after two repeated adsorption cycles are depicted in 4 (0 C m2), CO2 (14.3 × 10−40 C m2)), kinetic diameters (CH4 (3.8 Å), CO2 (3.3 Å)) and different boiling points ((CH4 (−161 °C), CO2 (−78 °C))). Described differences may influence the maximal adsorption uptake of gases.Compounds UPJS-7′′, UPJS-8′′ and UPJS-9′′ were also tested as gas adsorbents for carbon dioxide and methane at high pressures. Obtained CO−1 (2.99 wt%) for UPJS-7′′, 0.45 mmol g−1 (1.98 wt%) for UPJS-8′′ and 0.39 mmol g−1 (1.72 wt%) for UPJS-9′′ at 30 °C and 1 bar. A similar trend of adsorbed amounts was also observed for CO2 adsorption isotherms measured at 20 °C and 1 atm, described above. With increasing pressure, an increase in the amount of adsorbed CO2 was observed. The maximal adsorption uptake and corresponding adsorption capacities were 3.72 mmol g−1 (16.4 wt%) for UPJS-7′′, 3.31 mmol g−1 (14.6 wt%) for UPJS-8′′ and 3.07 mmol g−1 (13.5 wt%) for UPJS-9′′ at 30 °C and 20 bar. For repeated measurements, the branches of adsorption isotherms did not overlap and in the second repetition cycle, the samples adsorbed lower amounts of carbon dioxide compared to the first run. As was described in Section 2.5 Gas adsorption measurements above, the samples at repeated measurements were activated only at 30 °C for 8 hours. Observed results from CO2 high pressure adsorption suggest that CO2 molecules interact strongly with the frameworks of prepared compounds. A similar observation was demonstrated by the hysteresis loop on CO2 adsorption isotherms performed at low pressures.Compounds storage capacities of carbon dioxide at 1 bar and 30 °C were 0.68 mmol g4 with maximal storage capacity of 3.99 wt% (2.49 mmol g−1) for UPJS-7′′, 3.32 wt% (2.07 mmol g−1) for UPJS-8′′ and 2.86 wt% (1.78 mmol g−1) for UPJS-9′′ at 30 °C and 20 bar. The storage capacity of prepared compounds has a similar trend to carbon dioxide and decrease in order UPJS-7′′ > UPJS-8′′ > UPJS-9′′. It could be noted, that the amounts adsorbed for methane are approximately two times lower compared to carbon dioxide. In repeated CH4 adsorption measurements, isotherms do not show a significant change in adsorbed volume. This observation indicates that there is no significant interaction between the methane molecules and the polymeric frameworks. Interestingly, the compound does not adsorb CH4 at low pressures. The compounds start to adsorb methane up to 0.3 bar for UPJS-7′′, UPJS-8′′ and compound UPJS-9′′ at pressure up to 0.8 bar. On the adsorption isotherms, a breakthrough occurs at a pressure of about 2 bar, where the adsorbed amount of methane is significantly increased. The measured isotherms indicate a possible transformation of the polymeric skeletons, which was confirmed for compound UPJS-9 by HE-PXRD measurements. Measured adsorption results indicate the absence of specific adsorption sites for CH4 at the surface of prepared AE-MOFs.Methane was also used as a probe molecule for high pressure adsorption and measured isotherms are shown in 2 and CH4 adsorption measurements were performed for two compounds containing MTB linker. Nickel(ii) MOF with composition {[Ni4(μ6-MTB)2(μ2-H2O)4(H2O)4]·10DMF·11H2O}n (UPJS-3)20 adsorbed 3.29 wt% (2.05 mmol g−1) of CH4 and 18.05 wt% (4.1 mmol g−1) of CO2 and lead(ii) compound with formula {[Pb4(μ8-MTB)2(H2O)4]·5DMF·H2O}n (UPJS-5)20 adsorbed 3.05 wt% (1.9 mmol g−1) of CH4 and 19.81 wt% (4.5 mmol g−1) of CO2 at 30 °C and 21 bar. From the above results, it can be concluded that all prepared AE-MOFs show the same or slightly increased value of methane adsorption capacity and slightly reduced carbon dioxide adsorption capacity.According to the literature data, the high pressure CO2(DOBDC) ;46 Ca2(AZBZ-TC)(H2O)(DMF) 47 and Ba2(TMA)(NO3)(DMF) .48In the literature, only few MOF compounds containing alkaline earth metals on which the adsorption properties at high pressures and approximately the same conditions have been studied. As an example for comparison could be Mg4.ii) compounds UPJS-6 and UPJS-7, were prepared using different reaction temperatures. Compound UPJS-6 represents the low-temperature nonporous phase and compound UPJS-7 as the high temperature form exhibited CaF2 framework topology with an open structure. Compounds UPJS-8 and UPJS-9 also exhibit porosity with different pore dimensionality. The framework stability of prepared materials was investigated using thermogravimetric analysis and high energy powder X-ray diffraction. The thermal stability of prepared compounds ranged at high temperature 500–530 °C (exception UPJS-6), as a result of strong ionic interaction between AE ions and carboxylates of MTB linker. Moreover, HE-PXRD measurements also confirmed the stability of the frameworks and a phase transition was observed in compound UPJS-9. After the solvent exchange process of as-synthesized samples, materials were thermally activated under vacuum and tested as adsorbents for different probe molecules. Activated materials displayed small surface areas and moderate carbon dioxide storage capacities at low and high pressures, comparable with other MTB-containing compounds. Moreover, CO2 adsorption isotherms showed hysteresis at low pressure, respectively reduced adsorbed amount of carbon dioxide at repeated high pressure measurements, indicating an interaction of CO2 molecules with the frameworks. The results of methane adsorption measurements showed that the compounds do not adsorb CH4 at low pressures  and with increasing pressure the adsorbed volume increase. From observed results, it can be concluded that the compounds can be used in the separation process of methane and CO2 at low pressures. Moreover, compounds displayed increased adsorbed volume of hydrogen with a sorption capacity of 3.7–1.8 wt% at −196 °C and 800 Torr, which can be explained by the presence of coordinatively unsaturated sites in the MOF's frameworks.We have prepared and characterized four novel alkaline earth metal–organic frameworks. Structural analysis showed that compounds form 3D frameworks consisting of MTB linker and EA ions, which form different clusters with different SBU. Two different calcium(There are no conflicts to declare.RA-010-D0RA05145D-s001RA-010-D0RA05145D-s002


Coxsackievirus A16 (CVA16) causes hand, foot and mouth disease in infants and young children. However, no vaccine or anti-viral agent is currently available for CVA16. Here, the functions and working mechanisms of two CVA16-specific neutralizing monoclonal antibodies (MAbs), 9B5 and 8C4, are comprehensively investigated. Both 9B5 and 8C4 display potent neutralization in vitro and prophylactic and therapeutic efficacy in a mouse model of CVA16 infection. Mechanistically, 9B5 exerts neutralization primarily through inhibiting CVA16 attachment to cell surface via blockade of CVA16 binding to its attachment receptor, heparan sulfate, whereas 8C4 functions mainly at the post-attachment stage of CVA16 entry by interfering with the interaction between CVA16 and its uncoating receptor SCARB2. Cryo-EM studies show that 9B5 and 8C4 target distinct epitopes located at the 5-fold and 3-fold protrusions of CVA16 capsids, respectively, and exhibit differential binding preference to three forms of naturally occurring CVA16 particles. Moreover, 9B5 and 8C4 are compatible in formulating an antibody cocktail which displays the ability to prevent virus escape seen with individual MAbs. Together, our work elucidates the functional and structural basis of CVA16 antibody-mediated neutralization and protection, providing important information for design and development of effective CVA16 vaccines and antibody therapies. Coxsackievirus A16 (CVA-16) causes hand, food and mouth disease in infants and young children, however, currently there are neither anti-virals nor vaccines available for treatment. Here, Zhang et al. characterize two CVA16-specific neutralizing monoclonal antibodies, 9B5 and 8C4. They provide evidence for their prophylactic and therapeutic application in vivo in mice and perform CryoEM to show that both antibodies target different epitopes on the viral capsid. Enterovirus genus within the Picornaviridae family2, is one of the major causative agents of hand, foot and mouth disease (HFMD) prevalent in infants and young children6. CVA16 infection may result in mild and self-limiting symptoms7 as well as severe clinical outcomes such as encephalitis, myocarditis, pneumonitis, and even death10. In addition, CVA16 often co-circulates with other HFMD-causing agents such as human enterovirus A71 (EV71), coxsackievirus A6 (CVA6), and coxsackievirus A10 (CVA10), leading to co-infection and viral genetic recombination13, which make it more challenging to prevent and control HFMD as a whole.Coxsackievirus A16 (CVA16), a member of the 14. The viral genome encodes a large polyprotein precursor, which is subsequently processed into structural protein P1 and nonstructural proteins P2 and P314. P1 can be further cleaved by a viral protease to yield capsid subunit proteins VP0, VP1, and VP3, among which VP0 may undergo autocleavage to produce VP2 and VP414. CVA16 prepared from infected cell cultures is present mainly in two particle forms, including the mature virion  containing infectious viral RNA genome and the empty particle  lacking viral RNA genome17. The capsids of CVA16 full particle and empty particle are structurally similar, both made of 60 copies of protomers arranged in symmetry, however, each protomer in the full particle consists of four subunits, including VP1, VP2, VP3, and VP4, whereas the one in the empty particle comprises VP1, VP3, and uncleaved VP017. For most EVs, their mature virions are in the native/compact state, whereas their empty particles appear relatively expanded20. However, empty particles in compact state have also been reported for CVA16 and hepatitis A virus21.Like other enteroviruses (EVs), CVA16 is a nonenveloped virus of ~30 nm in diameter with a single-stranded, positive-sense RNA genome of ~7.4 kb in length packaged in a protein shell termed capsid22, and human scavenger receptor B2  as its uncoating receptor23. Upon receptor binding or biochemical treatment, CVA16 mature virions may transform to an uncoating intermediate state, termed the “135S-like particle” or “A-particle”, with typical structural features such as an expanded capsid, loss of pocket factor, and an enlarged two-fold opening25.CVA16 interacts with its host receptors to gain entry into susceptible cells. Specifically, CVA16 utilizes cell surface heparan sulfate (HS) glycosaminoglycans as its attachment receptor4. Neutralizing antibodies play a critical role in anti-viral protective immunity26. It is hence important to identify CVA16-specific neutralizing antibodies and determine their functionality, binding epitopes, and working mechanisms, which in turn may aid the development of anti-CVA16 vaccines and therapeutics. Previous studies have demonstrated that passive transfer of neutralizing antisera induced by CVA16 vaccine candidates, such as inactivated whole virus vaccines or recombinant virus like particles, could effectively protect mice against lethal CVA16 challenge29. A recent report showed that two CVA16-specific neutralizing monoclonal antibodies (MAb), namely 14B10 and NA9D7, displayed therapeutic efficacy in a mouse model of CVA16 infection17. A number of CVA16 neutralizing antibody epitopes have been identified through peptide ELISA screens or structural studies30. However, it remains elusive how antibodies targeting these epitopes/sites exert neutralization and protection. Especially, whether and how neutralizing antibodies affect CVA16 virus interacting with its receptors has not been explored.Thus far, neither preventive vaccines nor therapeutic drugs for CVA16 have been licensed for human useIn this work, we comprehensively characterize two CVA16-specific mouse neutralizing MAbs, designated 9B5 and 8C4, respectively. Both 9B5 and 8C4 antibodies display potent prophylactic and therapeutic efficacy in a mouse model of CVA16 infection. Mechanistic studies reveal that 9B5 and 8C4 target non-overlapping epitopes on the CVA16 viral capsid and adopt distinct mechanisms to exert neutralization. These two MAbs are compatible in formulating an antibody cocktail with increased neutralizing potency and, more importantly, able to prevent virus escape seen with individual antibodies. Our findings may have important implications for CVA16 vaccine and antibody development.31, did not exhibit any neutralization in any of the tested concentrations  and were then screened for neutralization against the parental virus CVA16/SZ05. Finally, three stable hybridoma mono-clones  showing neutralizing activity were obtained Fig. . Based oons Fig. . Isotypions Fig. . Antibod32 was highly effective in neutralizing EV71/G082  assay. Briefly, biotinylated CVA16/SZ05 viral particles were immobilized onto the sensors and then allowed to interact with different concentrations of individual MAbs. As shown in Fig. Next, we assessed whether the three MAbs competed with each other in binding CVA16 particles by performing a competition BLI assay. In this assay, immobilized CVA16/SZ05 viral particles were pre-incubated with kinetics buffer (reference) or the first antibody and then allowed to bind the second MAb in the presence of the first MAb . Binding signals of the second MAb were calculated and shown in Fig. 28. Besides individual MAbs, a combination of 8C4 and 9B5 at a ratio of 1:1 was also included in the in vivo protection studies, considering that the two antibodies recognize non-overlapping epitopes on viral capsid and thus can be used together. For the prophylaxis study, groups of naïve 1-day-old ICR mice were injected with PBS, 10 mg/kg of anti-CVA16 MAbs 8C4 or 9B5, the combination of 8C4 and 9B5 (10 mg/kg of each antibody), or 10 mg/kg of the IgG control antibody 2H231. Twenty-four hour later, the mice were challenged with CVA16/MAV. Survival and clinical scores were recorded daily and were depicted in Fig. The protective effects of MAbs 8C4 and 9B5 were assessed in a previously reported mouse model of CVA16 infection, which was based on the mouse-adapted strain CVA16/MAVTo evaluate the therapeutic efficacy of the anti-CVA16 MAbs, groups of 2-day-old ICR mice were infected with CVA16/MAV and 1 day later treated with PBS, 10 mg/kg of MAb 8C4, 10 mg/kg of MAb 9B5, or the 8C4 + 9B5 combination (10 mg/kg of each MAb). Survival and clinical scores were monitored daily Fig. . Mice giTo confirm the in vivo efficacy of the anti-CVA16 MAbs, both prophylactic and therapeutic tests were repeated and similar results were obtained  or the cells were treated with the MAbs at different time points after virus attachment at 4 °C (post-attachment). Then, the cells were analyzed for relative viral RNA levels at 6 h post-infection (hpi). As shown in Fig. Next, we examined whether 8C4 or 9B5 could inhibit CVA16 attachment, the first step of the viral entry process. Briefly, CVA16 viral particles were pre-incubated with the MAbs before binding to cooled RD cells at 4 °C, and after washing, RNA of CVA16 particles bound to cells was determined by quantitative RT-PCR. As shown in Fig. 22. We performed pulldown assays with heparin-conjugated agarose beads to determine whether 9B5 or 8C4 pre-treatment could block CVA16 binding to heparin. As shown in Fig. It has been previously shown that CVA16 binds soluble HS and utilizes cell surface HS glycosaminoglycans as its attachment receptor33. To test whether pre-treatment with 8C4 or 9B5 could block CVA16 binding to SCARB2 receptor, we carried out a competition BLI assay with recombinant human SCARB2 luminal domain fused with human IgG Fc (SCARB2-Fc). Briefly, immobilized CVA16/SZ05 viral particles were pre-incubated with kinetics buffer (reference), the IgG control antibody 2H231, MAb 8C4, or 9B5 and then allowed to interact with SCARB2-Fc in the presence of the antibody. Binding signals of SCARB2-Fc were recorded and depicted in Fig. 35, therefore prevent receptor-mediated virus uncoating.Human SCARB2 has been previously reported to play a critical role in the uncoating of EV71 and CVA16To further investigate the structural basis of CVA16 neutralization by 9B5 MAb, we performed cryo-EM study on the CVA16–9B5 Fab complex. SDS-PAGE analysis of the purified CVA16 sample revealed the presence of VP0, VP1, VP2 and, VP3 capsid proteins, with VP0 being more abundant than VP2   and the expanded state (~170 Å) Å) Fig. . The cha Å) Fig. , which i39. Both heavy and light chains of 9B5 Fab are involved in the antibody binding to CVA16 VP1 protein. Detailed analysis showed that the heavy chain complementarity-determining region 3 (HCDR3) and framework 3 (HFR3) of 9B5 bind to the BC loop (residues G99 and D104) of VP1, while the light chain CDR3 (LCDR3) contacts the VP1 EF (residue R166) and HI (residue K242) loops  on each protomer, and the 9B5 heavy and light chains contribute 51.3% and 48.7% of the overall binding interactions, respectively 40 into our C1 structure  and K242 (HI loop) of VP1 could form salt bridges or hydrogen bond with residues D93 and W92 in LCDR3 of 9B5, respectively  Fig. , and the2 on viral capsid, and the 8C4 heavy and light chains contribute 56.2% and 43.8% of the interface, respectively (Supplementary Table It appears that 8C4 Fab binds only to the native mature virion (C1) and compact empty particle (C2), rather than the expanded empty particle (C3) Fig. . DetaileIn the CVA16-8C4 C2 structure, the conformation of 8C4 binding site on the capsid is well maintained to accommodate the 8C4 Fab, despite both of the antibody and capsid protein display a very slight upward movement relative to the counterparts in the CVA16-8C4 C1 model , while the mutants from well #2 at 1/102 to 1/105 dilutions. Thus, 8C4-resistant mutants from well #1 had lower fitness than those from well #2 and wild-type CVA16. Sequence analysis of escape mutants showed that all (8/8 plaques) of the 8C4-selected isolates from well #1 harbored a V159F mutation in VP2, while those from well #2 carried a K69Q mutation in VP2  or T100A + T103A (6/8 plaques) double mutations in VP1  that target distinct epitopes42. However, a previous study on CVA16 indicated that formalin-treated CVA16 empty particle may exist in compact state16. In the current study, we found that the CVA16–9B5 C2 structure has a capsid radius (162.3 Å) similar to that (161.4 Å) of the compact mature virion in the C1 structure but does not contain viral RNA genome inside the capsid, and also presents closed, but not open, two-fold channels , indicating that the viral particle in the CVA16–9B5 C2 structure is indeed compact empty particle. A similar conclusion can also be drawn based on our CVA16–8C4 C2 map. Collectively, these data demonstrate the existence of naturally occurring compact empty particle of CVA16. Interestingly, we found that 9B5 and 8C4 exhibit different binding patterns to the three forms of CVA16 particles, including compact mature virion, compact empty particle, and expanded empty particle. Specifically, 9B5 is able to bind all three particle forms, whereas 8C4 recognizes only the compact particles (including mature virion and compact empty particle) but not the expanded empty particle  under selective pressure of individual MAb   Fig. . Howeverne) Fig. , demonstne) Fig. , and theIn summary, our study clearly defines the function and working mechanism of two groups of CVA16-specific neutralizing MAbs that target the five-fold and three-fold vertexes of the capsid, and reveals the existence of naturally occurring CVA16 compact empty particle that highly resembles the CVA16 mature virion in overall capsid structure and antigenic property. Our work also demonstrates the 9B5/8C4 antibody cocktail displays increased neutralization potency and acquires a unique ability to prevent virus escape seen with individual antibodies. These findings should have important implications for design and development of anti-CVA16 vaccines and therapies.The mouse studies were approved by the Institutional Animal Care and Use Committee at the Institut Pasteur of Shanghai. The mice were kept in the specific pathogen free animal facility with controlled temperature (20–26 °C), humidity (40–70%), and lighting conditions (12 h light/12 h dark cycle).28. EV71 strain EV71/G082 has been described previously44. All viruses were titrated by the 50% tissue culture infectious dose (TCID50) assay.Vero and rhabdomyosarcoma (RD) cells were grown in DMEM  with 5% fetal bovine serum (FBS). Mouse SP2/0 cells were grown in RPMI 1640 medium (Gibco) with 10% FBS. CVA16 strains used in this study include CVA16/SZ05 and a mouse-adapted strain CVA16/MAVg for 5 h. The resulting pellet was resuspended in 0.15 M PBS buffer and centrifuged to remove insoluble material. Next, the supernatant was further purified by 10–50% sucrose gradient ultra-centrifugations at 270,000 × g for 3 h. The fractions containing virus antigens were pooled and concentrated using Amicon Ultra 100 K filters . The final CVA16 virion sample was analyzed by SDS-PAGE and quantified by Bradford assay.To obtain CVA16 viral particles, Vero cells were grown to 80% confluency and then infected with CVA16/SZ05 at a multiplicity of infection of 0.01. At 2 days post-infection (dpi), culture supernatant was collected, and the cells were lysed in 0.15 M PBS buffer with 1% NP-40 and then clarified by centrifugation. Next, the culture supernatant and the clarified cell lysate were precipitated with 10% polyethylene glycol (PEG) 8000 and 200 mM NaCl. After high-speed centrifugation, the pellet was collected and resuspended in 0.15 M PBS buffer. After centrifugation to remove insoluble material, the supernatant was subjected to 20% sucrose cushion ultra-centrifugations at 112,700 × To generate SCARB2 protein, DNA fragment encoding the luminal domain of human SCARB2 (residues V28 to L433) was cloned into a modified pcDNA3.4 vector with an N-terminal IL-10 signal sequence and C-terminal human IgG1 Fc and His tag using ClonExpress II One Step Cloning Kit , yielding plasmid pcDNA3.4-SCARB2-hFc. The plasmid was then transfected into HEK 293F suspension cells using Polyethylenimine . After 5 days of culture, the culture supernatant was harvested by centrifugation and subjected to affinity purification using Ni-NTA resin  to obtain His-tagged SCARB2-hFc fusion protein.31 and used as a negative control in this study. MAb D5 is an antibody against EV7144.MAb 2H2 is an IgG antibody against SARS-CoV-228 in aluminum adjuvant . About 2 weeks after the third immunization, one mouse was injected intravenously with 10 μg of inactivated CVA16 antigen28 in PBS. Three days after the boost, splenocytes were taken from the mouse and fused with SP2/0 myeloma cells using PEG 1450 , followed by HAT (Sigma) selection for 10 days. Next, hybridoma supernatants were screened by neutralization assay with CVA16/SZ05 as described below. Positive hybridomas were cloned 2–4 times to generate monoclonal stable cell lines. Antibody isotypes were measured using SBA ClonotypingTM System/HRP ELISA kit  according to manufacturer’s protocol. Heavy and light chain variable region sequences were determined using the 5’ RACE System  or mouse Ig-primer set  according to the manufacturer’s protocols and then analyzed using the IgBLAST tool45.Adult female BALB/c mice  were immunized intraperitoneally (i.p.) three times at 2-week intervals with 4 µg/dose of inactivated CVA16 antigen46. Fabs were obtained by papain digestion of anti-CVA16 MAbs and then purified using Protein G column and Sephacryl S-100 column  according to our previously described protocols47.MAbs were purified from ascites using protein G agarose resin 4FF  according to our previously described protocol50) of CVA16/SZ05, CVA16/GX08, CVA16/MAV, or EV71/G082 in 96-well plates, followed by incubation at 37 °C for 1 h. Next, 100 µl/well  of RD cells were added to the plates and incubated at 37 °C. After about 3 days, the cultures were observed for CPE, and cell viability was then measured using CellTiter-Glo 2.0 assay kit  according to the manufacturer’s instructions. Luminescence measurements are expressed as relative luminescence units (RLU). Percent neutralization was calculated by the following equation: 100 × (RLU of the sample − RLU of the virus control wells) / (RLU of untreated cells − RLU of the virus control wells). For each MAb, its IC50  was calculated using GraphPad Prism software by nonlinear regression.Neutralizing activities of hybridoma supernatants and purified MAbs against CVA16 or EV71 were determined by micro-neutralization assay. Briefly, 50 µl/well of undiluted hybridoma culture supernatants or serially diluted purified MAbs were mixed with 50 µl/well  and then purified using Zeba™ spin desalting column (Thermo Fisher Scientific) according to the manufacturer’s protocols. The virus-binding affinity of the MAbs was determined by BLI assay in an Octet® RED96 System . Briefly, biotinylated CVA16/SZ05 viral particles were immobilized onto streptavidin biosensors . Next, these biosensors were transferred into wells containing a series of diluted individual antibody samples to permit virus-antibody association and then transferred into dissociation buffer . Equilibrium dissociation constants (KD) were calculated using Octet data analysis software v11.0 .For antibody competition assay, CVA16/SZ05 viral particles-coated sensors were transferred into wells containing buffer (control) or 15 µg/ml of the first MAb (the competitor antibody) for 500 s. The sensors were then moved into wells containing the second MAb alone  or the antibody mixture (15 µg/ml of the first MAb plus 15 µg/ml of the second MAb) for 500 s. Octet data analysis software was used to calculate the binding level of the second MAb.28. Pregnant female Institute of Cancer Research (ICR) mice (outbred model) were purchased from Vital River Laboratory Animal Technology company.The protective efficacy of anti-CVA16 MAbs was assessed in a mouse model of CVA16 infection based on the mouse-adapted strain CVA16/MAV31. After 24 h, the mice were i.p. infected with 1875 TCID50 of CVA16/MAV strain.For the prophylactic experiment, groups of naïve 1-day old ICR mice were i.p. injected with PBS, 10 mg/kg of anti-CVA16 MAbs (8C4 or 9B5), a mixture of both 8C4 and 9B5 (10 μg/g of each MAb), or 10 mg/kg of control MAb 2H250 of CVA16/MAV strain. After 24 h, the mice were i.p. injected with PBS, 10 mg/kg of MAb 8C4, 10 mg/kg of MAb 9B5, or a mixture of both 8C4 and 9B5 (10 mg/kg of each MAb). For the prophylactic and therapeutic assays, all infected mice were observed daily for survival and clinical score for 14 days. Clinical scores were graded as follows: 0, healthy; 1, reduced mobility; 2, limb weakness; 3, limb paralysis; 4, death.For the therapeutic assay, groups of 2-day-old ICR mice were i.p. injected with 1875 TCID48. Briefly, for pre-attachment inhibition assay, 1000 TCID50 of CVA16/SZ05 was mixed with 1 μg of each of the MAbs, and the virus-antibody mixtures were cooled on ice and then added to pre-chilled RD cells cultured in 24-well plates, followed by incubation at 4 °C for 1 h to permit viral attachment. The cells were then washed twice with chilled PBS, and fresh DMEM with 1% FBS was added and incubated at 37 °C.Time of addition assay was performed according to our previously described protocol50 of CVA16/SZ05 was added to pre-chilled RD cells and allowed to adsorb for 1 h at 4 °C. Next, the cells were washed twice with cold PBS and then incubated with fresh medium at 37 °C for 0 or 0.5 h to permit viral entry. Next, fresh media supplemented with 1 μg of individual MAbs was added to the wells, and the cells were re-incubated at 37 °C. For both assays, culture supernatant and RD cells were harvested together 6 h after infection and subjected to RNA isolation using TRIzol reagent (Invitrogen). cDNA was synthesized from the RNA using PrimeScript RT reagent Kit  and then subjected to real-time PCR analysis using SYBR Premix Ex Taq kit (Takara) with LightCycler® 480 II system  according to the manufacturer’s protocols. CVA16-specific primers were as follows: forward primer, 5′-ATCCAGTAAGGATCCCAGACT-3′; reverse primer, 5′-GATTTGCATAGTGGAGAGCAG-3′. β-actin primers were as follows: forward primer, 5′-GGACTTCGAGCAAGAGATGG-3′; reverse primer, 5′-AGCACTGTGTTGGCGTACAG-3′. Data were analyzed using the 2−ΔΔCt method with β-actin as the internal control.For post-attachment inhibition assay, 1000 TCID6 TCID50 of CVA16/SZ05 was incubated with various amounts  of each of the MAbs at 37 °C for 1 h. The mixtures were cooled on ice, then added to pre-chilled RD cells in 24-well plates and allowed to adsorb for 1 h at 4 °C. The cells were washed twice with cold PBS and then subjected to RNA isolation using TRNzol Universal reagent . cDNA was synthesized and subjected to real-time PCR analysis with CVA16-specific and β-actin-specific primers as described above. Data were analyzed using the 2−ΔΔCt method with β-actin as the internal control.In total, 1.0 × 107 TCID50 (500 μl) of CVA16/SZ05 was incubated with various amounts  of each of the MAbs at room temperature for 1 h. The virus-MAb mixtures were then mixed with 20 μl of heparin-agarose beads (Sigma), followed by incubation at room temperature for 1 h under gentle rotation. The beads were collected by centrifugation at 835 × g for 3 min, washed with PBS twice and resuspended in SDS loading buffer, followed by western blotting analysis with rabbit anti-CVA16-VP0 polyclonal antibody as primary antibody (1: 1000 dilution) and goat anti-rabbit IgG–HRP (sigma) as secondary antibody .In total, 1.0 × 10Biotinylated CVA16/SZ05 viral particles were coated onto streptavidin biosensors. Next, these biosensors were moved into wells containing buffer (control) or 15 µg/ml of each of the MAbs for 500 s. The sensors were then moved into wells containing SCARB2-hFc protein alone  or the MAb and SCARB2-hFc mixture (15 µg/ml of MAb plus 180 µg/ml of SCARB2-hFc) for 1000 s. Octet data analysis software was used to calculate the binding level of SCARB2-hFc.To prepare immune complexes, CVA16 virion and 9B5 or 8C4 Fab were incubated at a molar ratio of 1:120 for 20 min at room temperature. An aliquot of 3 µl of CVA16–9B5 or CVA16–8C4 complex was placed onto a plasma-cleaned holey carbon grids  or a continuous ultrathin carbon film covered lacey carbon grid , respectively. The grids were blotted and plunged into liquid nitrogen-cooled liquid ethane with a Mark IV Vitrobot (Thermo Fisher Scientific).−/Å2 on the specimen. For CVA16–8C4 complex, each frame was exposed for 0.1 s, and the total exposure time was 3 s, leading to a total accumulated dose of ~50 e−/Å2. Defocus values for both complexes ranged from −0.5 to −2.0 μm (Supplementary Table Cryo-EM movies of the samples were collected on a Titan Krios transmission electron microscope (Thermo Fisher Scientific) operated at an accelerating voltage of 300 kV, using FEI TEM user interface 2.15.3. The movies were recorded using a K3 Summit direct electron detector (Gatan) in counting mode (yielding a pixel size of 1.1), in an automatic manner using EPU 2.11 software (Thermo Fisher Scientific). For CVA16–9B5 complex, each frame was exposed for 0.05 s, and the total exposure time was 3 s, leading to a total accumulated dose of ~38 e50, and CTF parameters were determined using CTFFIND4.1.851 before further data processing. Unless otherwise described, the data processing was mainly performed in Relion 3.150.For each dataset, the motion correction of image stack was performed using the embedded module of Motioncor2 in Relion 3.152. After further 3D classification of the dataset into four classes, we obtained three better classes revealing distinct configurations. After CTF refinement, Bayesian polishing, and refinement, these three classes were subsequently refined to the CVA16–9B5 C1, C2, and C3 maps from 3122, 3967, and 3564 particles at the resolution of 2.90 Å, 3.35, and 3.80 Å, respectively. The overall resolutions for all of the cryo-EM maps in this study were determined based on the gold-standard criterion using a Fourier shell correlation of 0.14353.For the CVA16–9B5 dataset 16 as the initial model for the C1 state of both CVA16–9B5 and CVA16–8C4, the natural empty CVA16 structure (PDB: 5C9A)16 for C2 state, and the CVA16 empty particle structure (PDB: 6LHC)17 for the C3 state. In the meanwhile, we built the homology models of 9B5 and 8C4 Fab through the SWISS-MODEL webserver55. We then combined the model and flexibly refined the model against the corresponding cryo-EM map utilizing Rossetta 201756, then Phenix 1.10.157. Subsequently, to improve the fitting between model and map, we performed real-space refinement using COOT 0.8.358, then Phenix for the last round of flexible fitting of the entire complex. The final atomic models were validated by phenix.molprobity 1.10.1-215559. The validation statistics of the atomic models were summarized in Supplementary Table To build the atomic model for CVA16–9B5 and CVA16–8C4 maps, we used the corresponding cryo-EM structures of CVA16 from previous studies as initial model for the virial particle portion60. Fab-virion interaction analysis including hydrogen bond, salt bridge prediction, and buried surface area calculation were carried out through PISA server61. Roadmaps were generated by RIVEM 4.3 62. Interaction surface analysis was conducted by using PDBePISA 1.4863.Figures were generated using UCSF Chimera 1.10.250 of wild-type CVA16/SZ05 was incubated with various concentrations of MAb 8C4, 9B5, or 8C4 + 9B5 cocktail for 1 h at 37 °C before infection of RD cells in 96-well plates at 37 °C. After 4 days, the culture supernatants and cells were harvested and frozen and thawed twice, and 30 µl of the samples (passage 1 [P1] virus) were incubated with their selecting antibodies  for 1 h at 37 °C prior to infection of fresh RD cells. After 3 days, 30 µl of the resultant P2 virus was subjected to another round of selection with their selecting MAbs  for 1 h at 37 °C, yielding P3 virus. The resulting escape mutants were purified by plaque purification in RD cells overlaid with 0.4% low-melting point agarose (Promega) and 1% FBS in DMEM. For each MAb-selected mutant, 10 plaques were picked at 3 dpi and amplified in RD cells in 24-well plates. Total cellular RNA was extracted using TRIzol reagent (Invitrogen) and cDNA was the synthesized using M-MLV reverse transcriptase . The capsid protein P1-coding region was amplified by PCR and then sequenced.To screen escape mutants, 100 TCIDAll statistical analyses were performed using GraphPad Prism version 8.Further information on research design is available in the Supplementary informationPeer Review FileReporting Summary


Given the promising activity and tolerability of FOLFIRINOX as a second-line treatment for advanced biliary tract cancer (BTC), it can be an attractive first-line treatment option as well.This is a single-arm, open-label, multicenter phase II study to evaluate the safety and efficacy of FOLFIRINOX as a first-line treatment for patients with advanced BTC. Primary endpoint was progression-free survival (PFS), and the secondary endpoints included overall survival (OS), tumor response and safety. This study defined primary endpoint might be met when the lower limit value of 80% confidence interval [CI] of the median PFS ≥ 6.0 months.Between June 2016 and March 2020, 35 BTC patients  including 26 unresectable and 9 recurrent disease were enrolled. After a median follow-up of 13.9 months, the median PFS and OS were 7.4  and 14.7  months, respectively. Complete response was achieved in 1 (2.9%) and partial response in 10 (28.6%), giving an objective response rate of 31.4% and disease control rate of 74.3%. Major grade 3–4 adverse events included neutropenia (54.3%), leukopenia (34.4%), febrile neutropenia (17.1%), thrombocytopenia (8.6%), cholangitis (8.6%), anemia, nausea, diarrhea, and peripheral sensory neuropathy (2.9% each).FOLFIRINOX was well tolerable in patients with advanced BTC, however, this study did not meet the primary endpoint to conduct a phase III trial. Thus, further explorations are required to find a subset of patients and/or certain clinical scenario which might be beneficial from FOLFIRINOX. Biliary tract cancer (BTC) comprises heterogenous neoplasms derived from epithelial cells in the biliary system including either intrahepatic or extrahepatic cholangiocarcinoma and cancers of the gallbladder and ampulla of Vater. Although BTC accounts for 3% of all adult cancers, its incidence has been increasing worldwide, primarily due to a rise in intrahepatic cholangiocarcinoma . The proA combination therapy of gemcitabine and cisplatin (GC) is a current standard of care for advanced BTC because it provides longer survival without any substantial toxicity as compared with gemcitabine alone , 4. In aRecently, a survival benefit of FOLFOX  has been shown in patients with BTC refractory to the first-line gemcitabine-based chemotherapy . In addi3, absolute neutrophil count ≥ 2,000 /mm3, hemoglobin ≥ 9.0 g/dl, platelet count ≥ 100,000 /mm3, total bilirubin ≤ 1.2 times the upper limit of normal, aspartate aminotransferase and alanine aminotransferase levels ≤ 2.5 times the upper limit of normal, and creatinine clearance level ≥ 50 ml/min; 8) written informed consent from participants.Inclusion criteria were as follows: 1) histologically or cytologically proven BTC; 2) advanced or recurrent disease; 3) no prior chemotherapy or radiotherapy except for adjuvant chemotherapy, which had been completed at least 6 months before enrollment; 4) Eastern Cooperative Oncology Group (ECOG) performance status of 0–1; 5) age of 20–75 years; 6) At least one measurable lesion on computed tomography (CT) or magnetic resonance imaging (MRI) defined by Response Evaluation Criteria in Solid Tumors (RECIST) guideline (version 1.1); 7) adequate organ function, as indicated by white blood cell count ≤ 10,000 /mmThe exclusion criteria were as follows: 1) active concomitant malignancy; 2) hypersensitivity to iodine and gadolinium contrast; 3) brain metastasis; 4) blood infusion or hematopoietic growth factor use within 7 days before enrollment; 5) uridine diphosphate glucuronosyl transferase 1 family polypeptide A1 gene (UGT1A1) polymorphisms of homozygous UGT1A1*28 or UGT1A1*6 or heterozygous UGT1A1*6 and UGT1A1*28; 6) marked pleural effusion, or ascites active within 14 days before enrollment; 7) severe comorbidities, such as cardiac, hepatic or renal failure; 8) pulmonary fibrosis or intestinal pneumonitis; 9) grade 2 or higher peripheral sensory neuropathy; 10) active infection other than hepatitis B or C virus infection; 11) user of atazanavir sulfate; 12) pregnant or lactating woman; and 13) disqualified for trial judged by clinical investigators.2 over 2 h, irinotecan 180 mg/m2 over 90 min, leucovorin 400 mg/m2 over 2 h, fluorouracil 400 mg/m2 bolus infusion, each once daily on day 1, and fluorouracil 2,400 mg/m2 on day 1 over 46 h, every 2 weeks. During the cycle, if patients had a leucocyte > 10,000 ⁄mm3, neutrophil < 1,500 ⁄mm3, hemoglobin < 8.0 g/dL, platelet < 75,000 ⁄mm3, total bilirubin > 1.2 times of the upper limit, AST and ALT > 3 times of the upper limit, creatinine clearance < 50 ml/min, fever > 38℃, grade 3/4 of other non-hematological adverse events, treatment was temporary suspended and the next cycle was resumed after recovery from those toxicities with the reduced doses of each agent including an omission of the bolus fluorouracil infusion. The reduced doses were set at 85 mg/m2, 65 mg/m2, 50 mg/m2, and zero for oxaliplatin, 150 mg/m2, 120 mg/m2 and zero for irinotecan, and 1800 mg/m2 and 1200 mg/m2 for continuous fluorouracil infusion. The dose escalation and the use of primary prophylactic granulocyte colony stimulating factor (G-CSF) was not allowed according to the study protocol. The treatment was given until disease progression, unacceptable toxicity, discontinuation as decided by the investigators, or withdrawal of consent.We adopted an original, full-dose FOLFIRINOX regimen because at the time of study planning there was little evidence which supported modified regimen such as an omission of bolus-fluorouracil could ensure an improved safety without compromising efficacy of the treatment. Thus, patients initially received oxaliplatin 85 mg/mThe primary endpoint was PFS, and the secondary endpoints included OS, tumor response including objective response rate (ORR) and disease control rate (DCR), and adverse events. PFS was calculated from the start of treatment to the date of either disease progression or death or censored at last follow-up. OS was defined as the time from treatment initiation to final follow-up or until death from any cause. PFS and OS were calculated using the Kaplan–Meier method based on follow-up information, which was received until September 2021. ORR were evaluated with CT every 8 weeks until disease progression or death or censored at last follow-up using RECIST guidelines (ver. 1.1) . CarcinoA sample size of 35 patients was calculated to reject a null hypothesis defined as a median PFS of < 6.0 months and accept an alternative hypothesis defined as a median PFS ≥ 10.0 months, with a one-sided significance level of 0.10 and a power of 75%, assuming 5 years of recruitment and an additional 1.5 years of follow-up. In other words, FOLFIRINOX might be considered worthy of further evaluation in a randomized phase III trial if the lower limit value of 80% CI of the median PFS ≥ 6.0 months. All statistical analyses were performed using the program SAS .The study was an open-label, multi-center, prospective, single-arm, phase II study to evaluate the safety and efficacy of FOLFIRINOX as a first-line treatment for patients with advanced or recurrent BTC. The study protocol was approved by The Clinical Research Review Board of The University of Tokyo (2018006SP), and all patients provided written informed consent before enrollment. This study was conducted in accordance with the Declaration of Helsinki and the guidelines of Good Clinical Practice. And this study was registered in the University Hospital Medical Information Network (UMIN) Clinical Trial Registry (UMIN000020801) and Japan Registry of Clinical Trials .Between June 2016 and March 2020, 35 BTC patients were enrolled. All patients had histologically or cytologically proven advanced or recurrent BTC with at least one measurable lesion. Twenty-one patients (60.0%) had intrahepatic cholangiocarcinoma, 10 (28.6%) had extrahepatic cholangiocarcinoma, 2 (5.7%) had gallbladder cancer and 2 (5.7%) had ampulla of Vater cancer . The median relative dose intensities of oxaliplatin, irinotecan, bolus fluorouracil, infusional fluorouracil, and leucovorin were 62.8%, 60.2%, 13.1%, 76.5%, and 80.0%, respectively. Dose reduction and treatment delay occurred in 30 patients (85.7%). Neutropenia was the most frequent cause for both dose reduction and treatment delay .After a median follow-up of 13.9 months, the median PFS and OS were 7.4  and 14.7  months, respectively Fig. . The 12-As shown in Table A complete response was achieved in 1 2.9%), partial response in 10 (28.6%) and stable disease in 15 (42.9%), giving an ORR of 31.4% and DCR of 74.3%  at baseline, CA19-9 was reduced by more than 50% compared to pretreatment level in 9 patients (37.5%), which was associated with tumor response .All eligible patients were evaluated for toxicities. Grade 3 or 4 adverse events occurred in 31 patients (88.6%). As shown in Table n = 6), obstructive jaundice (n = 1) and biliary tract infection (n = 1), and an increased level of blood bilirubin (n = 1), all of which were unrelated to the study treatment. For patients with (n = 8) or without (n = 27) a biliary stent, febrile neutropenia was observed in 3 (37.5%) and 3 (11.1%), respectively and cholangitis was observed in 4 (50.0%) and 2 (7.4%), respectively.Seven episodes of febrile neutropenia accounted in 6 patients mainly during the first two cycles, and a total of 28 sessions of G-CSF was used in 14 patients (40.0%) to control febrile neutropenia as well as severe neutropenia. The G-CSF usage decreased as the number of cycles increased . Biliary tract-related events were reported in 8 patients, including cholangitis , unacceptable toxicity in 4 , withdrawal of consent in 2 (5.7%), or conversion surgery with curative intent after remarkable tumor shrinkage in 3 (8.6%). Twenty-five patients (71.4%) received second-line chemotherapy , and 14 patients (40.0%) received third-line chemotherapy . In total, four patients were converted to be surgical candidates after chemotherapy and they subsequently underwent surgery, resulting in R0 resection in 3. Notably, the surgical specimen revealed a pathologic complete response in one patient with initially metastatic intrahepatic cholangiocarcinoma.This prospective, multicenter phase II study showed FOLFIRINOX was well tolerable and potentially effective in the first-line treatment of advanced BTC, with a median PFS of 7.4  months and OS of 14.7  months, and an ORR and DCR of 31.4% and 74.3%, respectively. However, since this study did not meet the predefined criterion to conduct a phase III trial , further explorations are required to find a subset of patients and/or certain clinical scenario which might be beneficial from FOLFIRINOX.Since FOLFIRINOX significantly improved the survival in patients with metastatic pancreatic cancer , it had vs 5 months; p = 0.008) [Recent insights into genomic characterization of BTC leads to intensive investigations to develop a target therapy . Rationa= 0.008) . Thereaf= 0.008) , 14. In = 0.008) , 33. Mos= 0.008) , 35. How= 0.008) . With thFOLFIRINOX for pancreatic cancer is generally reserved to young patients with good performance status and less comorbidity in current clinical practice because of the concerns for safety. To date, modified regimen such as an omission of bolus fluorouracil or initial dose reduction of irinotecan has been recognized as a community-standard in patients with pancreatic cancer since it ensured the efficacy with an improved tolerability . In thisThis study has several limitations. First, our study is not a randomized, controlled trial included heterogenous patients in terms of origin of diseases with a relatively small sample size. Since this likely interferes with the interpretation of results, future studies will need to consider enrolling patients with specific origin of diseases to appreciate more significant results from homogenous populations and guide clinical practice with a disease location-specific approach. Second, our study required longer time to enroll patients than expected due to slow recruitment. During the study period, gene-panel tests using next-generation sequencing have been introduced into clinical practice in Japan, promoting a personalized precision medicine for BTC. Although gene-panel tests will drive a big paradigm shift in the treatment strategy for BTC, patients with actionable mutations or molecular targets are still limited, necessitating further explorations for novel cytotoxic treatment options. Thus, despite its inherent limitations, our study results would provide some evidence to the current treatment options for BTC.In conclusion, FOLFIRINOX was safe and potentially effective an option for first-line chemotherapy in patients with advanced BTC but further explorations are required to find a subset of patients and/or certain clinical scenario which might be beneficial from FOLFIRINOX.