Synthetic self-assembling ADDomer platform for highly efficient vaccination by genetically encoded multiepitope display

Charles Vragniau
Joshua C Bufton
Frédéric Garzoni
Emilie Stermann
Fruzsina Rabi
Céline Terrat
Mélanie Guidetti
Véronique Josserand
Matt Williams
Christopher J Woods
Gerardo Viedma
Phil Bates
Bernard Verrier
Laurence Chaperot
Christiane Schaffitzel
†
Imre Berger
Pascal Fender
Synthetic self-assembling ADDomer platform for highly efficient vaccination by genetically encoded multiepitope display
Vragniau et al., Sci. Adv. 2019; 5 : eaaw2853 25 September 2019 S C I E N C E A D V A N C E S | R E S E A R C H A R T I C L E 1 of 8 S T R U C T U R A L B I O L O G Y
Self-assembling virus-like particles represent highly attractive tools for developing next-generation vaccines and protein therapeutics. We created ADDomer, an adenovirus-derived multimeric protein-based self-assembling nanoparticle scaffold engineered to facilitate plug-and-play display of multiple immunogenic epitopes from pathogens. We used cryo-electron microscopy at near-atomic resolution and implemented novel, cost-effective, high-performance cloud computing to reveal architectural features in unprecedented detail. We analyzed ADDomer interaction with components of the immune system and developed a promising first-in-kind ADDomer-based vaccine candidate to combat emerging Chikungunya infectious disease, exemplifying the potential of our approach.

INTRODUCTION

Self-assembling protein-based nanoparticles are highly attractive tools for a broad range of biomedical applications, including vaccine development and cancer therapy (1)(2)(3)(4). Present in all kingdoms of life, they form supramolecular architectures with unique properties (4), including spontaneous self-organization from simple precursor protomers amenable to engineering. Moreover, the particle size is generally in the range of pathogens, notably viruses, which the immune system has evolved to strongly react against (5,6). Protein-based nanoparticles often adopt quasi-spherical shapes encapsulating a central cavity that can carry cargo, rendering them suitable to deliver drugs (3). Virus-like particles (VLPs) are made of many copies of identical building blocks resulting in highly repetitive surfaces, providing opportunities to display pathogen-derived epitopes. These are often oligopeptide sequences, on their own generally not immunogenic enough to elicit a strong immune response resulting in protection (5,6). However, if coupled to self-assembling protomers forming the VLP, then peptide epitopes can reach very high densities on the VLP, potentially able to trigger B cell receptor clustering and crosspresentation for facilitation of a strong immune response (5,7). Notably, VLPs can act as self-adjuvants, alleviating the need to supplement vaccine formulations with additional adjuvanting reagents that can have undesired side effects (8).

VLPs are intensely researched toward new and better vaccines and therapeutics against acute and chronic diseases (2,3,(9)(10)(11). An ideal VLP-based vaccine would be safe, free of nucleic acid contaminants, amenable to engineering, well produced recombinantly, easy to purify, available in large amounts, self-adjuvanting, and capable of eliciting a strong immune response to pathogen-derived antigens presented in a native conformation at high density on the particle surface. A major aspect is thermotolerance-many vaccines today require refrigeration, rendering storage and deployment dependent on a functioning cold chain, posing problems in remote or less affluent regions (12). Thus, thermostability would be a highly advantageous asset for any VLP vaccine.

Human adenoviruses are among the most widely used vectors in gene therapy (13). The icosahedral adenovirus capsid comprises distinct proteins arranged in hexons, pentons, and fiber extrusions. The penton-forming protomer of certain adenovirus serotypes can self-assemble into a symmetric, hollow dodecahedron adopted by 12 pentons comprising altogether 60 protomers (14), effectively representing a proteinaceous, nonenveloped VLP. We showed previously that human adenovirus serotype 3 (Ad3) dodecahedron can be produced in baculovirus-infected insect cells (15) and can retain the adenovirus-like ability to penetrate epithelial cells (16). We studied the structure and mechanism of the Ad3-derived dodecahedron, investigating its potential as a delivery system for DNA, proteins, and chemical compounds (17)(18)(19). Encouraged by these promising proof-of-concept results, we set out here to create a synthetic, engineered Ad3-derived stable dodecahedron-forming protomer for highly efficient multiepitope display and delivery toward a next-generation ADDomer VLP platform for vaccines and protein therapeutics.

RESULTS

ADDomer multiepitope display and delivery platform

Comparison of primary sequences of penton-forming protomers from many adenovirus serotypes revealed particularly two regions of high variability in length and sequence, called variable loop (VL) and arginine-glycine-aspartic acid (RGD) loop. For instance, VL comprises 20 amino acids in Ad3 while only 9 amino acids in Ad41. RGD loop plasticity is even more pronounced in Ad12, spanning 11 residues, in contrast to 74 residues in Ad2. The VL has been shown previously to accommodate an influenza-derived immunogenic eptitope (20). The RGD loop contains a conserved tripeptide motif (-RGD-) mediating integrin-based internalization into target cells (21). We reasoned that these regions, VL and RGD loop, given their polymorphism, would be ideally suited to accommodate one or several antigenic epitopes each. Therefore, we redesigned the protomerencoding gene adopting a "BioBrick" format (22) to facilitate multiple epitope insertions. We inspected the crystal structure of Ad3 dodecahedron (17) and identified suitable loci adjacent to secondary structure motifs flanking the loops. The RGD loop in wild-type Ad3 is considerably more extended than the VL. As the tripeptide is important for cell internalization, we decided to split the RGD loop into two sections before and after the RGD motif, which we kept unaltered. We inserted unique restriction sites to create three independent loci for epitope display, one in the VL and two in the RGD loop to facilitate insertion of synthetic DNAs encoding for antigenic epitopes of choice in the resulting plug-and-play multiepitope display platform, the ADDomer ( Fig. 1A and figs. S1 and S2). We expressed ADDomer using MultiBac, the baculovirus/insect cell system we developed for complex protein biologics (23), resulting in highly purified ADDomer (fig. S1) with excellent culture yields (50 mg from a 50-ml culture) and negligible nucleic acid or endotoxin contamination.

ADDomer structure and mechanism

Unlocking ADDomer to future structure-based design, we determined its molecular architecture by high-resolution cryo-electron microscopy (cryo-EM) (Fig. 1, B to H, and figs. S3 and S4). For parts of the process, we used public cloud resources, implementing image processing and refinement software for this purpose. Our cryo-EM structure, at up to 3.2-Å resolution, reveals ADDomer adopting the familiar dodecahedron, formed by 12 pentons in a quasi-spherical arrangement, with the RGD loop and VL densely decorating the particle surface (Fig. 1, B and C, fig. S3, and table S1). Our structure shows that the plasticity of the RGD loop and VL, essential for antigenic epitope presentation, is maintained in ADDomer (Fig. 1D).

Crystal structures of wild-type Ad3 dodecahedron with and without fiber peptide bound exist at lower resolution (3.8 and 4.8 Å, respectively) (17). Close inspection of our cryo-EM structure reveals notable differences. The fiber-binding cleft adopts a rearranged conformation, seemingly in between the geometries observed in the crystals of apo-and fiber-bound Ad3 dodecahedron (Fig. 1E) (17). The protomer represents a two-domain architecture, with a crown region comprising the VL and RGD loop abutting a jellyroll fold mediating multimerization (Fig. 1, D and F). In our cryo-EM structure, we observed additional density in between the crown and jellyroll domains, juxtaposed to four sulfur-containing amino acids (M131, M138, M458, and C539), consistent with a tetradentate coordination by a metal ion, possibly zinc or iron (Fig. 1F). We posit that a structural metal ion at this interdomain interface could be important for stabilizing this protein fold.

In the Ad3 dodecahedron crystal, a Ca 2+ ion occupied a central cavity lined by glutamates (E466) in -helices from different protomers (Fig. 1G). No corresponding density is observed by cryo-EM. The Ad3 crystallization conditions contained CaCl 2 , whereas we did not supplement Ca 2+ during ADDomer purification to occupy this site.

Consequently, glutamates E466 do not extend into the cavity, but the corresponding -helices are slightly shifted in the ADDomer.

A hallmark in the Ad3 crystal structure was strand-swapping between extended N-terminal regions of neighboring protomers, deemed essential to dodecahedron structural integrity (17). Unexpectedly, we did not observe this strand-swapping by cryo-EM ( Fig. 1H and  fig. S4C). Rather, in ADDomer, two neighboring protomers engage in hydrogen bonds in the vicinity of the twofold axis, extending from the S61 side chain to the peptide backbone of E59 and vice versa. We can exclude that the differences observed are due to ADDomer amino acid additions and substitutions within the VL and RGD loop, as they are in the crown domain and thus sterically too distant to have a bearing on the N-terminal domain, or the formation of the hitherto unobserved metal binding site, respectively.

Strand-swapping and Ca 2+ ions would conceivably stabilize the dodecahedron. We did not observe either of these features by cryo-EM and thus wondered about ramifications for ADDomer stability. Thermal shift assays evidenced a melting temperature of 54°C for ADDomer and no noticeable melting below 45°C ( fig. S5A). We challenged ADDomer integrity by storing for weeks at room temperature, freezing and thawing, and incubating at 45°C. Negative-stain EM revealed uniformly stable ADDomer ( fig. S5, B to E). Thus, we can conclude that AD-Domer maintains highly advantageous thermotolerance, begging the question what exactly the source of this remarkable stability may be.

ADDomer (30 nm) is within the size range (20 to 200 nm) of particles readily drained to lymph nodes, potentiating uptake by antigen-presenting cells and cross-presentation (5,6). We analyzed ADDomer uptake by human cells, including monocytes and monocytederived dendritic cells (MDDCs), confirming efficient internalization (Fig. 2, A and B). We next investigated lymph node distribution in mice injected with fluorescently labeled ADDomer (Fig. 2, C and D). Rapid draining to the right inguinal lymph node was observed irrespective of the mode of administration. No signal was found in the opposite left inguinal node serving as internal control. Mesenteric and axillary nodes evidenced rapid but not lasting signal (Fig. 2D). Our results underscore ADDomer capacity to drain rapidly to the nearest lymph node and to efficiently penetrate human cells, including antigen-presenting lymphocytes.

Engineering ADDomer to combat emerging infectious diseases

Infectious diseases continue to decimate populations worldwide, and among our means to combat this threat, vaccination is exceptionally powerful. Recent examples that dominated the media are Chikungunya and Zika infectious diseases, transmitted by mosquitoes originally confined to sub-Saharan Africa but increasingly spreading to the Northern Hemisphere. We used our plug-and-play setup to rapidly generate ADDomers displaying multiple copies of immunogenic epitopes from a range of human and livestock viral pathogens, testing the capacity of the insertion loci in the process ( fig. S6 and table S2). To assess the flexibility of the ADDomer to harbor large multiepitope insertions, we combined five immunogenic Gumboro-derived epitopes in tandem. Gumboro is caused by infectious bursal disease virus and affects poultry, causing significant economic shortfall, particularly in developing countries. ADDomer evidenced a remarkable plasticity for the insertion of the five tandem Gumboro-derived epitopes in both functionalized VL and RGD loop without compromising integrity ( fig. S6, A and B). Insertion in the VL evidenced a blurring of the penton edges, suggesting preferred orientations of the loop structure comprising the Gumboro epitopes. Notably, we successfully inserted Three evolutionary nonconserved segments (boxed) in the protomer were engineered by BioBrick design into exchangeable cassettes. One cassette (yellow) is located within the VL, while two cassettes, RGD1 (light green) and RGD2 (dark green), are located within the loop containing a functional RGD tripeptide sequence. Numbers indicated amino acid boundaries of BioBrick cassettes as well as VL and RGD loop. (B) Left: A 3.5-Å cryo-EM map of the ADDomer particle formed by 60 protomers. The rigid core is colored blue, and more flexible regions comprising the loops are colored cyan and green. Right: The corresponding atomic model is shown in gray. One penton (center) is highlighted, with individual protomers colored red, orange, green, cyan, and blue, respectively. a test epitope exceeding 200 amino acid residues in the RGD loop ( fig. S6B). Together, our data demonstrate full functionality of the insertion loci we engineered and convey that long sequences containing multiple epitopes can be efficiently displayed at high density on the ADDomer.

Notably, sera of most patients suffering from Chikungunya viral infection react with a specific linear peptide, E2EP3 (24). This major neutralizing epitope is located at the very N terminus of the viral E2 glycoprotein, comprising the first 18 amino acids ( Fig. 3A and table S2). We inserted E2EP3 into ADDomer to create a VLP vaccine candidate, ADDomer-tevCHIK, with the potential to combat this debilitating disease. To recapitulate the exposed conformation of E2EP3 in Chikungunya virus, we inserted a site for a highly specific protease [tobacco etch virus (TEV) NIa] right in front of the epitope, which, upon cleavage, generates a native-like conformation comprising an exposed N-terminal serine (Fig. 3A). Processing by TEV resulted in quantitative cleavage (Fig. 3B). Negative-stain EM confirmed that, impressively, the ADDomer dodecahedron remained intact notwithstanding multiple (60-fold) polypeptide backbone cuts (Fig. 3C). Immunization experiments compellingly validated our approach. Uncut ADDomer-tevCHIK did not yield E2EP3-specific immunoglobulin titers ( fig. S7). In contrast, quantitatively cleaved ADDomer-tevCHIK exposing native-like E2EP3 elicited very strong specific immunoglobulin G (IgG) response concomitant with barely detectable IgM (Fig. 3D and figs. S7 and S8), in excellent agreement with the immune reaction to Chikungunya viral infection. An unrelated nanoparticle scaffold made of polylactic acid harboring the same amount of CHIK peptide epitopes did not result in an immune response above background in mice, suggesting that the ADDomer scaffold itself may have an adjuvanting effect ( fig. S7).

DISCUSSION

In summary, we presented here ADDomer, our plug-and-play multiepitope display platform for antigenic epitopes from different origins, including emerging human viral pathogens or veterinary infectious diseases. We determined ADDomer architecture at near-atomic resolution by cryo-EM, enabled by a heterogeneous computation cluster and created and executed automatically with public cloud infrastructure. Our approach presents a novel, fast, and cost-effective general mechanism to quickly analyze and process cryo-EM data and sets the stage for comprehensive (re)design of the ADDomer particle, also to fine-tune self-adjuvanting potential by modulating sequence plasticity (25). ADDomer is easy to modify and produce in high yields using our recombinant system. It is remarkably thermostable, which could render deployment of ADDomer-based vaccines independent of a cold chain, representing a crucial asset. We demonstrate that ADDomer rapidly drains to lymph nodes and efficiently internalizes in immune blood cells. We inserted multiepitope sequences exceeding 200 amino acids without compromising ADDomer integrity, conveying that even folded domains or entire proteins could be displayed. ADDomer can be cleaved multiple times in loops without adverse effects, enabling native-like display of epitopes adopting exposed conformations, compellingly demonstrated here with Chikungunya E2EP3. Notably, modification of ADDomer is not restricted to the engineered loci. The fiber binding cleft could also be used to attach peptides and proteins, tagged with fiber peptide. Moreover, the N-terminal extension was shown to react with specific binder domains (19), conferring further capacity. Thus, ADDomer can accommodate hundreds of epitopes, same or different, providing vast opportunity for combinatorial display by a single particle ( fig. S9). Last, the central cavity within ADDomer could be used to encapsulate costimulatory molecules. We anticipate that a wide range of applications in biomedical research will benefit from this exceptionally versatile display platform.

MATERIALS AND METHODS

ADDomer design

We used the penton base protein from human Ad3 (GenBank Z29487) as a starting point and implemented a BioBrick format (22) to functionalize one locus within the VL and two loci in the RGD loop for facile and rapid custom DNA insertion (fig. S1). The insertion loci were flanked by unique restriction sites that were eliminated from elsewhere in the Ad3 penton base encoding gene. To preserve structural integrity of the penton base protomer, the restriction sites were designed in silico to be located outside of and adjacent to known secondary structure elements in the penton base protein. The resulting gene encoding for ADDomer was synthesized, codon-optimized for recombinant expression, and pasted into plasmid pACEBac1 (Geneva Biotech SARL, Switzerland) (26) using Bam HI and Hind III restriction sites, giving rise to pACEBac-ADDomer. The primary sequence of the encoded ADDomer is provided in fig. S2. In this study, pACEBac-ADDomer served as the backbone for inserting DNA encoding for selected epitopes into the designed insertion loci in the variable and RGD loops. Primary sequences of the inserted epitopes in this study are listed in table S2.

Expression and purification

pACEBac-ADDomer and epitope-displaying variants were expressed using the MultiBac baculovirus expression system we developed ( fig. S1) (23). Briefly, pACEBac-ADDomer and variant plasmids were inserted by Tn7 transposition into an engineered baculoviral genome, EMBacY, in DH10EMBacY cells (27). Transfection, baculovirus amplification, storage, and protein expression in Hi5 insect cell suspension cultures (50 ml) were performed following established protocols (27), resulting in exceptional yields ( fig. S1). The EMBacY baculoviral genome encodes a yellow fluorescent protein (YFP) marker. When the YFP signal reached a plateau, cells were harvested by centrifugation, and pellets were flash-frozen in liquid nitrogen and stored at −20°C. Pellets were resuspended in hypotonic lysis buffer [0.33× phosphate-buffered saline (PBS); 1 ml per 2.5 × 10 7 cells] supplemented by EDTA-free cOmplete protease inhibitor (Roche, Switzerland) and lysed by three cycles of freezing and thawing. Lysate was cleared from debris by centrifugation (6000g for 30 min), and the cleared supernatant was subjected to sucrose gradient (15 to 40%) centrifugation (15). Fractions containing ADDomer (30 to 40% sucrose) were pooled and dialyzed against buffer A [10 mM Hepes (pH 7.4) and 50 mM NaCl]. ADDomer was further purified by ion exchange chromatography using a High Q column (Bio-Rad) using buffer A and applying a linear salt gradient (50 to 500 mM NaCl). Fractions containing highly purified ADDomer (330 to 400 mM NaCl) were pooled and stored at ambient temperature or frozen (−80°C). For animal studies, ADDomer particles were further purified using Detoxi-Gel (Thermo Fisher Scientific) to remove endotoxins, followed by dialysis against PBS.

Following purification, absorbance spectroscopy measurement showed an OD 260nm (optical density at 260 nm)/OD 280nm ratio below 0.6, evidencing lack of nucleic acid contamination. Before injection into mice, endotoxin levels were quantified using Endosafe PTS (Charles River) evidencing 754 EU (endotoxin units)/mg for ADDomer-tevCHIK and 247 EU/mg for ADDomer, respectively. In our injections, we thus were below 30 EU, the common cutoff value for testing adjuvant properties of any recombinant antigen in a mouse model.

Thermostability measurements

Highly purified ADDomer sample was stored in PBS at room temperature. Thermal shift experiments were performed at an ADDomer concentration of 3 mg/ml in PBS, using a ThermoFluor assay (28). ADDomer sample was diluted to 1 mg/ml and aliquoted. Identical sample aliquots were stored for several weeks at room temperature, frozen at −20°C overnight followed by thawing, and incubated at 45°C . Scatter dot plot diagram depicts the outcome of the immunization experiments, revealing high individual IgG titers specific for Chikungunya E2E3P peptide in enzyme-linked immunosorbent assay (ELISA). Short horizontal line segments are geometric mean titers, and error bars represent the 95% confidence interval. Significant difference (****P < 0.0001) was determined between ADDomer-tevCHICK-and ADDomer-only immunized mice. for 1 hour, respectively. Sample integrity was assessed at each intervention by negative-stain EM ( fig. S5).

Negative-stain EM

Negative-stain EM was performed with purified ADDomer (0.1 to 0.5 mg/ml) dialyzed into 25 mM Hepes (pH 7.5), 150 mM NaCl, and 2 mM EDTA. Sample was stained with 3% uranyl acetate. Micrographs were recorded using a 200-kV FEI Tecnai T20 microscope with an Eagle 4k × 4k charge-coupled device camera.

Cryo-EM and data collection

Five microliters of purified ADDomer (0.1 mg/ml) was applied to glow-discharged holey carbon grids (Quantifoil, R 2/2 300 mesh). Samples were incubated on the grid for 2 s and blotted for 0.4 s at 90% relative humidity and 18°C inside a Leica EM GP, before plungefreezing in liquid ethane. Cryo-EM data were collected at 200 kV with a FEI Talos Arctica microscope equipped with a Gatan K2 direct electron detector and an energy filter using automated acquisition software (EPU). A total of 1060 dose-fractionated movies each containing 32 frames (0.25 s per frame) with an accumulated total dose of 42 e − /Å 2 were recorded at a nominal magnification of ×130,000 corresponding to a pixel size of 1.06 Å. Images were recorded with a defocus range of −0.8 to −3.2 m (table S1).

Image processing

Image processing was performed with the RELION 2.1 software package (29). The micrographs were motion-corrected using MotionCorr2 (30), and contrast transfer function (CTF) information was determined using ctffind4.1 (31). Together, 262 micrographs with CTF rings extending beyond 4.6 Å were selected for further processing. A total of 3600 particles were boxed using RELION autopicking software. After two-dimensional (2D) classification ( fig. S3C) and 3D classification with imposed icosahedral symmetry, initial 3D autorefinement led to a reconstruction of ~4.2-Å resolution. Further rounds of 3D classification/refinement were carried out on polished particles before using postprocessing for masking and automatic B factor sharpening. The resolution of the final map was determined to be 3.5 Å based on the Fourier shell correlation (FSC) 0.143 cutoff criterion ( fig. S3D) (32). Local resolution was calculated using local resolution estimation program in RELION ( fig. S3E).

Processing and 3D classification of ADDomer were performed using public cloud resources provided by the Oracle Cloud Infrastructure. Using this rather than an institutional cluster allowed a faster turnaround time of analysis. Because of the particular nature of the computational work carried out by the steps within RELION, a heterogeneous cluster was prepared with both central processing unit (CPU)-and graphical processing unit (GPU)-focused resources. A pipeline of workloads was created, allowing the work best suited to each compute type to use just the resources needed and to automatically release compute resources when no longer needed. This permitted parts of the workflow to use lower-cost CPU resources and to burst to GPU only for those parts that would benefit from GPU optimization, reducing overall cost while increasing total data throughput. The total cost for running the image processing pipeline on the cloud was approximately £200.

Model building and refinement

Homology modeling was performed using I-TASSER (33) using the human adenovirus Ad3 structure as template (17). The model was adjusted to fit into the map manually using COOT (34) before further iterative positional and B factor refinement in real space using Phenix Real-Space refinement software (35). Final adjustments were carried out in COOT before evaluating the model using MolProbity (36). Refinement statistics are summarized in table S1.

Cell culture and differentiation

HeLa cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (FCS) (Thermo Fisher Scientific) at 37°C under 5% CO 2 . Monocytes were purified by centrifugation on Ficoll (Sigma-Aldrich) for 20 min at 800g, and mononucleated cells were subsequently passed through EasySep magnetic beads (STEMCELL Technology). Monocyte purity was verified by flow-cytometry by means of CD14 labeling. MDDCs were generated by cultivating monocytes in RPMI 1640 FCS medium supplemented with granulocyte-macrophage colony-stimulating factor at 100 ng/ml and interleukin-4 at 25 ng/ml for 6 days. Differentiation to MDDCs was verified by FACS based on CD14/CD209 labeling.

Imaging

HeLa cells were grown on a glass coverslip and incubated for 1 hour at 37°C with unlabeled ADDomer at 20 g/ml. Subsequent to methanol fixation, rabbit serum raised against human Ad3 penton base protein, diluted 1:1000 in PBS, was applied, followed by Cy5-labeled goat anti-rabbit antibody (Jackson ImmunoResearch), diluted 1:500 in PBS. Nuclei were stained by the DNA dye 4′,6-diamidino-2-phenylindole.

Monocytes and MDDCs from healthy anonymous donors were obtained from the National Blood Service [Etablissement Francais du Sang (EFS), La Tronche] after written consent following approval by the EFS. ADDomer (1 mg/ml) was labeled with Alexa Flour 488 dye (Invitrogen) following the manufacturer's recommendations. Unbound dye was removed by extensive dialysis against PBS overnight. About 10 5 monocytes or MDDCs, respectively, were incubated for 1 hour at 37°C with 10 g of labeled ADDomer. Cells were applied to glass coverslips and analyzed using differential interference contrast and green fluorescence channels in an Olympus IX81 inverted microscope.

Biodistribution studies

ADDomer was labeled with Alexa Fluor 680 dye (Invitrogen) following the same procedure as above for Alexa Flour 488 labeling. Alexa Flour 680-labeled ADDomer sample was injected in 9-week-old female BALB/cJRj mice (Janvier Labs, France). Four control mice (no injection) were used for tissue autofluorescence quantifications. Mice were anesthetized (isoflurane/air, 4% for induction and 1.5% thereafter), and injections were performed either subcutaneously (n = 8; right flank; 100 l; 0.1 mg/ml) or intramuscularly (n = 8; right quadriceps; 12 l; 0.85 mg/ml). Mice were euthanized at either 1 hour (n = 4 per group) or 5 hours (n = 4 per group) after injection. Main lymph nodes (mesenteric, sciatic, axillary, inguinal, and brachial) were sampled and imaged using FLUOBEAM700 (Fluoptics, France) with 680-nm excitation and LP (Long Pass) 700-nm emission. Fluorescence radiance from each lymph node was quantified in relative light units per pixel per 100 ms.

Immune studies Initial test immunizations

Six-week-old female BALB/c mice were subcutaneously injected, and sera were collected and treated as described previously (37).

Immunizations were carried out with 10 g each of ADDomer, ADDomer-tevCHIK (uncut), or ADDomer-tevCHIK quantitively cleaved by TEV protease, respectively. Specific antibodies in sera were detected by enzyme-linked immunosorbent assay (ELISA) in duplicate using 96-well plates coated with CHIK peptide (0.1 g/ml). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG 1030-05 (SouthernBiotech) was used for detection [concentration of 0.1 g/ml in PBS with 1% bovine serum albumin (BSA)] for 1 hour at 37°C, supplemented with 100 l of tetramethylbenzidine (TMB) substrate (Becton Dickinson) per well. Reactions were stopped by adding H 2 SO 4 (0.2 N), and OD was measured in a microplate reader (Thermo Fisher Scientific) at 450 nm ( fig. S7).

ADDomer-tevCHIK immunization experiments

Ten B6D2F1 mice per group received twice (study days 1 and 15) subcutaneously 40 g per mouse of ADDomer (group 1) or quantitatively cleaved ADDomer-tevCHIK (group 2). Control mice were immunized twice with the buffer diluent. Mice were observed cage-side for clinical symptoms. Mice immunized with ADDomer (group 1) and ADDomer-tevCHIK (group 2) developed high serum IgG titers (<12,800) after the first and second immunization. Mice immunized with diluent did not induce ADDomer-specific IgG. IgM and IgG titers were <100 in serum samples collected before immunization. All mice immunized with ADDomer-tevCHIK sample developed CHIK-specific IgG after the first immunization (GMT, 2425; titer range, 400 to 6400). After the second immunization, IgG titers substantially increased (GMT, 19,400; titer range, 6400 to 51,200). Mice immunized with ADDomer (empty) and diluent did not develop CHIK-specific IgG titers. In contrast, only 1 of 10 mice immunized with ADDomer-tevCHIK developed detectable CHIK epitope-specific IgM after the first (titer, 1:50) and second (titer, 1:100) immunization. No CHIK-specific IgM could be determined in mice immunized with ADDomer or diluent.

Blood was collected by the facial vein technique at days 1, 14, and 35. Briefly, the mouse was gently and securely restrained in the operator's nondominant hand. The hairless freckle on the side of the jaw was located and pricked with a sharp lancet held by operator's free hand (sharp end of the lancet points at the far side of the mouse's face, at the base of the far ear, or at the base of the far side of the mouth). About four to seven drops of blood were collected in an Eppendorf tube, and the mouse was released into its cage. Blood samples were centrifuged twice in a bench centrifuge for 5 min at 10,000g at 4°C, and collected serum was stored below −18°C before analyses. An ELISA protocol was developed using streptavidin-coated plates and the biotinylated CHIK peptide (table S2) following established protocols (Thermo Fisher Scientific). Briefly, streptavidincoated plates were washed three times with 200 l of wash buffer [25 mM tris-HCl (pH 7.2), 150 mM NaCl, 0.1% BSA, and 0.05% Tween 20]. Next, 100 l of biotinylated CHIK peptide dissolved in dimethyl sulfoxide and diluted in PBS to a final concentration of 2.5 g/ml in PBS was added to each well. Plates were incubated for 2 hours on a plate shaker at room temperature. After extensive washing, twofold serially diluted serum samples were added (100 l per well). Plates were incubated for 1 hour with occasional shaking at room temperature. After three additional washing steps, 100 l of goat anti-mouse IgM or HRP-conjugated IgG antibody (diluted 1:2000; Sigma-Aldrich), respectively, was added to each well, and plates were incubated for 45 min at room temperature. Plates were again extensively washed before adding 100 l of color substrate (TMB) per well, followed by shaking. The color reaction was stopped with H 2 SO 4 (0.2 N), and OD was measured at 450 nm. To determine serum IgM or IgG titers, a cutoff value was defined as mean absorption value of negative blank samples plus three times the SD.

In vivo experimentation statement

In vivo experiments were carried out in France at the Institute for Advanced Biosciences (Grenoble) and the Institute of Biology and Chemistry of Proteins (Lyon). These laboratories are certified by the French Ministry of Research and Education Superior and regularly monitored by the French Departmental Directorate for the Protection of Population (approval numbers C 69 123 0303 and C38 516 10001). Immunization studies were carried out at the Institute of Virology, Biomedical Research Centre, Slovak Academy of Sciences (Bratislava). Animal care was in compliance with the standard operation procedures of the Institute of Virology and the European Convention for the Protection of Vertebrate Animals used for experimental and other scientific purposes (ETS 123) with approval from the Veterinary State Administration, Slovak Republic (Statna veterinarna a potravinova sprava Slovenskej republiky), and institutional ethical committee. All animal experiments were carried out according to the rules in force and in the respect of the ethics of the animal experimentation.

Statistics

Statistical significance was determined by calculating SDs following standard mathematical formulae (unpaired t test with Welch's correction of log-transformed titer values).

SUPPLEMENTARY MATERIALS

Fig. 1 .
1Cryo-EM structure of ADDomer. (A) ADDomer design.

(C) Side view of a penton formed by five protomers. Flexible VL and RGD loop are drawn in dashed lines. (D) Closeup view of the RGD loop and VL in ADDomer. Residues 156 to 170 in VL and 314 to 366 in RGD loop in the BioBrick format maintain their flexibility, essential for functionalization by epitope insertion. (E) Superimposition of the fiberbinding region in the ADDomer cryo-EM structure (cyan) with apo Ad3 [Protein Data Bank (PDB) ID: 4AQQ; marine blue] and fiber peptide-bound Ad3 (PDB ID: 4AR2; magenta) crystal coordinates. The fiber peptide (PDB ID: 4AR2) is drawn in a ball-and-stick representation. (F) Individual ADDomer protomer is shown in a side view (left) with a putative metal-binding cluster boxed in between the crown (top) and jellyroll fold (bottom) domains. Zoomed-in views of the boxed region (right) depict the atomic model in the EM density contoured at two different levels,  = 4 (blue) and  = 10.5 (red). Four juxtaposed sulfurs (yellow spheres) likely coordinate a metal ion. (G) Left: Central channel of an ADDomer penton. Right: Ca 2+ coordination as seen previously in the Ad3 penton base protein crystal structure (PDB ID: 4AR2) is not observed. The helices fencing the central channel are rearranged, resulting in a different conformation of E466. (H) Interface between two pentons (A1 and A2) in the ADDomer (cyan). The zoomed-in view depicts the boxed region with the corresponding Ad3 crystal structure (magenta) superimposed. Domain swapping is not observed in the ADDomer.Instead, this interface is stabilized by mutual hydrogen bonds between S61 side chains and E59 peptide backbones from neighboring protomers (right).

Fig. 2 .
2ADDomer internalizations. (A) HeLa cells incubated with ADDomer. 4′,6-diamidino-2-phenylindole-stained nuclei are colored blue. ADDomer detected by punctuatespecific immunofluorescence is colored red. Scale bar, 20 m. (B) Human monocytes (Mo; left) and MDDCs (right) were incubated with Alexa Fluor 488-labeled ADDomer (green). Efficient uptake of ADDomer was observed by in all cell types tested. Scale bars, 20 m. (C) Lymph node biodistribution of Alexa Fluor 680-labeled ADDomer in mice.Ex vivo imaging of the main lymph nodes from one representative mouse 5 hours after subcutaneous injection (10 g) in the right leg is depicted in a schematic drawing (left). Specific fluorescence signal was quantified (for 100 ms), and intensities (in arbitrary units) are plotted in spectral colors (bottom). (D) Ex vivo quantification (for 100 ms) of ADDomer in isolated lymph nodes from a cohort of mice (n = 8) is shown in a bar diagram. Fluorescence was measured at 1 and 5 hours, respectively, after intramuscular or subcutaneous injection of Alexa Fluor 680-labeled ADDomer (10 g). Bar color coding is as follows: dark blue, intramuscular at 1 hour; brown, intramuscular at 5 hours; light blue, subcutaneous for 1 hour; red, subcutaneous for 5 hours; black, control (noninjected) mice (n = 5). SDs are shown as error bars. Axi, axillary; Bra, brachial; Sci, sciatic; Ing fpr, inguinal; Mes, mesenteric lymph nodes; RLU, relative light unit per pixel.

Fig. 3 .
3ADDomer immunizations. (A) Top: Chikungunya envelope protein E2, composed of domains A, B, and C, is shown in a schematic representation. The major neutralizing epitope E2EP3 (red) comprises the N terminus of E2 with the amino acid sequence NH2-STDKDNFNVYKATRPYLAH. Bottom: This specific configuration of E2E3P was mimicked in ADDomer-tevCHIK as depicted by inserting the E2EP3 sequence (in red), preceded by a TEV protease cleavage site (orange) into the BioBrick cassette in VL of the ADDomer protomer. Cleavage by the protease exposes E2EP3 in a native-like configuration as shown schematically. (B) SDS-polyacrylamide gel electrophoresis section showing purified ADDomer-tevCHIK before and after cleavage by TEV protease. Molecular weight marker sizes are marked (left). Appearance of two bands (black triangles) indicates quantitative cleavage. (C) Representative negative-stain EM images of TEV-cleaved ADDomer-tevCHIK reveals stable dodecamers. Scale bar, 30 nm. (D) This TEV-cleaved sample was used in immunization experiments in mice (n = 10 per group)

New routes and opportunities for modular construction of particulate vaccines: Stick, click, and glue. K D Brune, M Howarth, Front. Immunol. 91432K. D. Brune, M. Howarth, New routes and opportunities for modular construction of particulate vaccines: Stick, click, and glue. Front. Immunol. 9, 1432 (2018).

Engineering virus-like particles as vaccine platforms. K M Frietze, D S Peabody, B Chackerian, Curr. Opin. Virol. 18K. M. Frietze, D. S. Peabody, B. Chackerian, Engineering virus-like particles as vaccine platforms. Curr. Opin. Virol. 18, 44-49 (2016).

Towards protein-based viral mimetics for cancer therapies. U Unzueta, M V Céspedes, E Vázquez, N F Miralles, R Mangues, A Villaverde, Trends Biotechnol. 33U. Unzueta, M. V. Céspedes, E. Vázquez, N. F. Miralles, R. Mangues, A. Villaverde, Towards protein-based viral mimetics for cancer therapies. Trends Biotechnol. 33, 253-258 (2015).

Bioengineering strategies for protein-based nanoparticles. D Diaz, A Care, A Sunna, Genes. 9370D. Diaz, A. Care, A. Sunna, Bioengineering strategies for protein-based nanoparticles. Genes 9, E370 (2018).

Vaccine delivery: A matter of size, geometry, kinetics and molecular patterns. M F Bachmann, G T Jennings, Nat. Rev. Immunol. 10M. F. Bachmann, G. T. Jennings, Vaccine delivery: A matter of size, geometry, kinetics and molecular patterns. Nat. Rev. Immunol. 10, 787-796 (2010).

Virus-induced humoral immunity: On how B cell responses are initiated. F Zabel, T M Kündig, M F Bachmann, Curr. Opin. Virol. 3F. Zabel, T. M. Kündig, M. F. Bachmann, Virus-induced humoral immunity: On how B cell responses are initiated. Curr. Opin. Virol. 3, 357-362 (2013).

TLR signaling in B-cell development and activation. Z Hua, B Hou, Cell. Mol. Immunol. 10Z. Hua, B. Hou, TLR signaling in B-cell development and activation. Cell. Mol. Immunol. 10, 103-106 (2013).

Comparative safety of vaccine adjuvants: A summary of current evidence and future needs. N Petrovsky, Drug Saf. 38N. Petrovsky, Comparative safety of vaccine adjuvants: A summary of current evidence and future needs. Drug Saf. 38, 1059-1074 (2015).

Production of virus-like particles for vaccines. J Fuenmayor, F Gòdia, L Cervera, N. Biotechnol. 39J. Fuenmayor, F. Gòdia, L. Cervera, Production of virus-like particles for vaccines. N. Biotechnol. 39, 174-180 (2017).

Viruses as vaccine vectors for infectious diseases and cancer. S J Draper, J L Heeney, Nat. Rev. Microbiol. 8S. J. Draper, J. L. Heeney, Viruses as vaccine vectors for infectious diseases and cancer. Nat. Rev. Microbiol. 8, 62-73 (2010).

Active immunotherapy for chronic diseases. M F Bachmann, P Whitehead, Vaccine. 31M. F. Bachmann, P. Whitehead, Active immunotherapy for chronic diseases. Vaccine 31, 1777-1784 (2013).

Improving cold chain systems: Challenges and solutions. A Ashok, M Brison, Y Letallec, Vaccine. 35A. Ashok, M. Brison, Y. LeTallec, Improving cold chain systems: Challenges and solutions. Vaccine 35, 2217-2223 (2016).

Adenovirus: The first effective in vivo gene delivery vector. R G , Hum. Gene Ther. 25R. G. Crystal, Adenovirus: The first effective in vivo gene delivery vector. Hum. Gene Ther. 25, 3-11 (2014).

The relationship between the soluble antigens and the virion of adenovirus type 3. II. Identification and characterization of an incomplete hemagglutinin. E Norrby, Virology. 30E. Norrby, The relationship between the soluble antigens and the virion of adenovirus type 3. II. Identification and characterization of an incomplete hemagglutinin. Virology 30, 608-617 (1966).

Adenovirus dodecahedron, a new vector for human gene transfer. P Fender, R W Ruigrok, E Gout, S Buffet, J Chroboczek, Nat. Biotechnol. 15P. Fender, R. W. Ruigrok, E. Gout, S. Buffet, J. Chroboczek, Adenovirus dodecahedron, a new vector for human gene transfer. Nat. Biotechnol. 15, 52-56 (1997).

Synthesis, cellular localization, and quantification of penton-dodecahedron in serotype 3 adenovirus-infected cells. P Fender, A Boussaid, P Mezin, J Chroboczek, Virology. 340P. Fender, A. Boussaid, P. Mezin, J. Chroboczek, Synthesis, cellular localization, and quantification of penton-dodecahedron in serotype 3 adenovirus-infected cells. Virology 340, 167-173 (2005).

The structural basis for the integrity of adenovirus Ad3 dodecahedron. E Szolajska, W P Burmeister, M Zochowska, B Nerlo, I Andreev, G Schoehn, J P Andrieu, P Fender, A Naskalska, C Zubieta, S Cusack, J Chroboczek, PLOS ONE. 746075E. Szolajska, W. P. Burmeister, M. Zochowska, B. Nerlo, I. Andreev, G. Schoehn, J. P. Andrieu, P. Fender, A. Naskalska, C. Zubieta, S. Cusack, J. Chroboczek, The structural basis for the integrity of adenovirus Ad3 dodecahedron. PLOS ONE. 7, e46075 (2012).

The use of adenovirus dodecahedron in the delivery of an enzymatic activity in the cell. B Sumarheni, P Gallet, Fender, Biotechnol. Res. Int. 5030589Sumarheni, B. Gallet, P. Fender, The use of adenovirus dodecahedron in the delivery of an enzymatic activity in the cell. Biotechnol. Res. Int. 2016, 5030589 (2016).

In vivo delivery of antigens by adenovirus dodecahedron induces cellular and humoral immune responses to elicit antitumor immunity. A Villegas-Mendez, M I Garin, E Pineda-Molina, E Veratti, J A Bueren, P Fender, J L Lenormand, Mol. Ther. 18A. Villegas-Mendez, M. I. Garin, E. Pineda-Molina, E. Veratti, J. A. Bueren, P. Fender, J. L. Lenormand, In vivo delivery of antigens by adenovirus dodecahedron induces cellular and humoral immune responses to elicit antitumor immunity. Mol. Ther. 18, 1046-1053 (2010).

Influenza recombinant vaccine: Matrix protein M1 on the platform of the adenovirus dodecahedron. A Naskalska, E Szolajska, L Chaperot, J Angel, J Plumas, J Chroboczek, Vaccine. 27A. Naskalska, E. Szolajska, L. Chaperot, J. Angel, J. Plumas, J. Chroboczek, Influenza recombinant vaccine: Matrix protein M1 on the platform of the adenovirus dodecahedron. Vaccine 27, 7385-7393 (2009).

The adenovirus type 3 dodecahedron's RGD loop comprises an HSPG binding site that influences integrin binding. E Gout, G Schoehn, D Fenel, H Lortat-Jacob, P Fender, J. Biomed. Biotechnol. 541939E. Gout, G. Schoehn, D. Fenel, H. Lortat-Jacob, P. Fender, The adenovirus type 3 dodecahedron's RGD loop comprises an HSPG binding site that influences integrin binding. J. Biomed. Biotechnol. 2010, 541939 (2010).

Engineering BioBrick vectors from BioBrick parts. R P Shetty, D Endy, T F KnightJr, J. Biol. Eng. 25R. P. Shetty, D. Endy, T. F. Knight Jr., Engineering BioBrick vectors from BioBrick parts. J. Biol. Eng. 2, 5 (2008).

The multibac baculovirus/insect cell expression vector system for producing complex protein biologics. D Sari, K Gupta, D B Thimiri-Govinda-Raj, A Aubert, P Drncová, F Garzoni, D Fitzgerald, I Berger, Adv. Exp. Med. Biol. 896D. Sari, K. Gupta, D. B. Thimiri-Govinda-Raj, A. Aubert, P. Drncová, F. Garzoni, D. Fitzgerald, I. Berger, The multibac baculovirus/insect cell expression vector system for producing complex protein biologics. Adv. Exp. Med. Biol. 896, 199-215 (2016).

Early neutralizing IgG response to Chikungunya virus in infected patients targets a dominant linear epitope on the E2 glycoprotein. Y W Kam, F.-M Lum, T.-H Teo, W W L Lee, D Simarmata, S Harjanto, C.-L Chua, Y.-F Chan, J.-K Wee, A Chow, R T P Lin, Y.-S Leo, R Le Grand, I.-C Sam, J.-C Tong, P Roques, K.-H Wiesmüller, L Rénia, O Rötzschke, L F P Ng, EMBO Mol. Med. 4Y. W. Kam, F.-M. Lum, T.-H. Teo, W. W. L. Lee, D. Simarmata, S. Harjanto, C.-L. Chua, Y.-F. Chan, J.-K. Wee, A. Chow, R. T. P. Lin, Y.-S. Leo, R. Le Grand, I.-C. Sam, J.-C. Tong, P. Roques, K.-H. Wiesmüller, L. Rénia, O. Rötzschke, L. F. P. Ng, Early neutralizing IgG response to Chikungunya virus in infected patients targets a dominant linear epitope on the E2 glycoprotein. EMBO Mol. Med. 4, 330-343 (2012).

Assessing sequence plasticity of a virus-like nanoparticle by evolution toward a versatile scaffold for vaccines and drug delivery. Y Lu, W Chan, B Y Ko, C C Vanlang, J R Swartz, Proc. Natl. Acad. Sci. U.S.A. 112Y. Lu, W. Chan, B. Y. Ko, C. C. VanLang, J. R. Swartz, Assessing sequence plasticity of a virus-like nanoparticle by evolution toward a versatile scaffold for vaccines and drug delivery. Proc. Natl. Acad. Sci. U.S.A. 112, 12360-12365 (2015).

MultiBac: Expanding the research toolbox for multiprotein complexes. C Bieniossek, T Imasaki, Y Takagi, I Berger, Trends Biochem. Sci. 37C. Bieniossek, T. Imasaki, Y. Takagi, I. Berger, MultiBac: Expanding the research toolbox for multiprotein complexes. Trends Biochem. Sci. 37, 49-57 (2012).

MultiBac: Multigene baculovirus-based eukaryotic protein complex production. C Bieniossek, T J Richmond, I Berger, 5.20.1-5.20.26Curr. Protoc. Protein Sci. 51C. Bieniossek, T. J. Richmond, I. Berger, MultiBac: Multigene baculovirus-based eukaryotic protein complex production. Curr. Protoc. Protein Sci. 51, 5.20.1-5.20.26 (2008).

A thermal stability assay can help to estimate the crystallization likelihood of biological samples. F Dupeux, M Röwer, G Seroul, D Blot, J A Márquez, Acta Crystallogr. Sect. D Biol. Crystallogr. 67F. Dupeux, M. Röwer, G. Seroul, D. Blot, J. A. Márquez, A thermal stability assay can help to estimate the crystallization likelihood of biological samples. Acta Crystallogr. Sect. D Biol. Crystallogr. 67, 915-919 (2011).

RELION: Implementation of a Bayesian approach to cryo-EM structure determination. S H W Scheres, J. Struct. Biol. 180S. H. W. Scheres, RELION: Implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519-530 (2012).

Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. X Li, P Mooney, S Zheng, C R Booth, M B Braunfeld, S Gubbens, D A Agard, Y Cheng, Nat. Methods. 10X. Li, P. Mooney, S. Zheng, C. R. Booth, M. B. Braunfeld, S. Gubbens, D. A. Agard, Y. Cheng, Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat. Methods 10, 584-590 (2013).

Accurate determination of local defocus and specimen tilt in electron microscopy. J A Mindell, N Grigorieff, J. Struct. Biol. 142J. A. Mindell, N. Grigorieff, Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334-347 (2003).

Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. P B Rosenthal, R Henderson, J. Mol. Biol. 333P. B. Rosenthal, R. Henderson, Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721-745 (2003).

I-TASSER server for protein 3D structure prediction. Y Zhang, BMC Bioinf. 940Y. Zhang, I-TASSER server for protein 3D structure prediction. BMC Bioinf. 9, 40 (2008).

Coot: Model-building tools for molecular graphics. P Emsley, K Cowtan, Acta Crystallogr. Sect. D Biol. Crystallogr. 60P. Emsley, K. Cowtan, Coot: Model-building tools for molecular graphics. Acta Crystallogr. Sect. D Biol. Crystallogr. 60, 2126-2132 (2004).

Real-space refinement in Phenix for cryo-EM and crystallography. P V Afonine, B K Poon, R J Read, O V Sobolev, T C Terwilliger, A Urzhumtsev, P D Adams, Acta Crystallogr. Sect. D Biol. Crystallogr. 74P. V. Afonine, B. K. Poon, R. J. Read, O. V. Sobolev, T. C. Terwilliger, A. Urzhumtsev, P. D. Adams, Real-space refinement in Phenix for cryo-EM and crystallography. Acta Crystallogr. Sect. D Biol. Crystallogr. 74, 531-544 (2018).

MolProbity: All-atom structure validation for macromolecular crystallography. V B Chen, W B Arendall, Iii , J J Headd, D A Keedy, R M Immormino, G J Kapral, L W Murray, J S Richardson, D C Richardson, Acta Crystallogr. Sect. D Biol. Crystallogr. 66V. B. Chen, W. B. Arendall III, J. J. Headd, D. A. Keedy, R. M. Immormino, G. J. Kapral, L. W. Murray, J. S. Richardson, D. C. Richardson, MolProbity: All-atom structure validation for macromolecular crystallography. Acta Crystallogr. Sect. D Biol. Crystallogr. 66, 12-21 (2010).

Cutting edge: New chimeric NOD2/TLR2 adjuvant drastically increases vaccine immunogenicity. V Pavot, N Rochereau, J Rességuier, A Gutjahr, C Genin, G Tiraby, E Perouzel, T Lioux, F Vernejoul, B Verrier, S Paul, J. Immunol. 193V. Pavot, N. Rochereau, J. Rességuier, A. Gutjahr, C. Genin, G. Tiraby, E. Perouzel, T. Lioux, F. Vernejoul, B. Verrier, S. Paul, Cutting edge: New chimeric NOD2/TLR2 adjuvant drastically increases vaccine immunogenicity. J. Immunol. 193, 5781-5785 (2014).

Traffic of poly(lactic acid) nanoparticulate vaccine vehicle from intestinal mucus to sub-epithelial immune competent cells. C Primard, N Rochereau, E Luciani, C Genin, T Delair, S Paul, B Verrier, Biomaterials. 31C. Primard, N. Rochereau, E. Luciani, C. Genin, T. Delair, S. Paul, B. Verrier, Traffic of poly(lactic acid) nanoparticulate vaccine vehicle from intestinal mucus to sub-epithelial immune competent cells. Biomaterials 31, 6060-6068 (2010).

A purified truncated form of yeast Gal4 expressed in Escherichia coli and used to functionalize poly(lactic acid) nanoparticle surface is transcriptionally active in cellulo. S Legaz, J.-Y Exposito, A Borel, M P Candusso, S Megy, R Montserret, V Lahaye, C Terzian, B Verrier, Protein Expr. Purif. 113S. Legaz, J.-Y. Exposito, A. Borel, M. P. Candusso, S. Megy, R. Montserret, V. Lahaye, C. Terzian, B. Verrier, A purified truncated form of yeast Gal4 expressed in Escherichia coli and used to functionalize poly(lactic acid) nanoparticle surface is transcriptionally active in cellulo. Protein Expr. Purif. 113, 94-101 (2015).

In silico modeling and immunoinformatics probing disclose the epitope based peptide vaccine against zika virus envelope glycoprotein. M M A K Shawan, H A Mahmud, M Hasan, A Parvin, N Rahman, S M B Rahman, Indian J. Pharm. Biol. Res. 2M. M. A. K. Shawan, H. A. Mahmud, M. Hasan, A. Parvin, N. Rahman, S. M. B. Rahman, In silico modeling and immunoinformatics probing disclose the epitope based peptide vaccine against zika virus envelope glycoprotein. Indian J. Pharm. Biol. Res. 2, 44-57 (2014).

Variation of a newcastle disease virus hemagglutinin-neuraminidase linear epitope. S.-H Cho, H.-J Kwon, T.-E Kim, J.-H Kim, H.-S Yoo, S.-J Kim, J. Clin. Microbiol. 46S.-H. Cho, H.-J. Kwon, T.-E. Kim, J.-H. Kim, H.-S. Yoo, S.-J. Kim, Variation of a newcastle disease virus hemagglutinin-neuraminidase linear epitope. J. Clin. Microbiol. 46, 1541-1544 (2008).

Development of a multi-mimotope peptide as a vaccine immunogen for infectious bursal disease virus. Y.-S Wang, H.-J Fan, Y Li, Z.-L Shi, Y Pan, C.-P Lu, Vaccine. 25Y.-S. Wang, H.-J. Fan, Y. Li, Z.-L. Shi, Y. Pan, C.-P. Lu, Development of a multi-mimotope peptide as a vaccine immunogen for infectious bursal disease virus. Vaccine 25, 4447-4455 (2007).

The structure of the human adenovirus 2 penton. C Zubieta, G Schoehn, J Chroboczek, S Cusack, Mol. Cell. 17C. Zubieta, G. Schoehn, J. Chroboczek, S. Cusack, The structure of the human adenovirus 2 penton. Mol. Cell 17, 121-135 (2005).

Acknowledgments: We thank J.-P. Molens for help with monocyte purification and maturation and W. Burmeister for assistance with structure analysis. We are particularly grateful to L. Signor for mass spectrometric analyses. Acknowledgments: We thank J.-P. Molens for help with monocyte purification and maturation and W. Burmeister for assistance with structure analysis. We are particularly grateful to L. Signor for mass spectrometric analyses;

Kleman for help with cell imaging. F Lacroix, J.-P , F. Lacroix and J.-P. Kleman for help with cell imaging;

We acknowledge access and support of the Wolfson Bioimaging Facility funded by the Wolfson Foundation and University of Bristol and GW4 Facility for High-Resolution Electron Cryo-Microscopy funded by the Wellcome Trust. B Ferko, GRALR Shattock, GRALS Bahrami, GRALD Fenel, GRALG Schoehn, GRALJ Mantel, GRALM Walker, GRALA Richardson, GRALSupport, GRALANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). BBSRC (BB/R000484/1). This work received support from the European Commission (FP7 HEALTH ComplexINC. contract no. 279039), the FINOVI Foundation, the GRAL valorization program, the CNRS prematuration program, the EPSRC Innovative Future Vaccine Manufacturing Hub (EP/R013764/1), and BrisSynBio (a BBSRC/EPSRC Research Centre for synthetic biology at the University of BristolB. Ferko, R. Shattock, and S. Bahrami for help with designing and interpreting immunizations; and D. Fenel, G. Schoehn, J. Mantel, M. Walker, and A. Richardson for EM support. Funding: This work used the platforms of the Grenoble Instruct Center (ISBG; UMS 3518 CNRS-CEA-UJF-EMBL) with support from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). We acknowledge access and support of the Wolfson Bioimaging Facility funded by the Wolfson Foundation and University of Bristol and GW4 Facility for High-Resolution Electron Cryo-Microscopy funded by the Wellcome Trust (202904/Z/16/Z and 206181/Z/17/Z) and BBSRC (BB/R000484/1). This work received support from the European Commission (FP7 HEALTH ComplexINC; contract no. 279039), the FINOVI Foundation, the GRAL valorization program, the CNRS prematuration program, the EPSRC Innovative Future Vaccine Manufacturing Hub (EP/R013764/1), and BrisSynBio (a BBSRC/EPSRC Research Centre for synthetic biology at the University of Bristol;

ADDomer is a registered trademark owned by Imophoron Ltd. The authors declare that they have no other competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Coordinates and EM data have been deposited at the public databases EMDB (EMD-0198) and PDB (PDB ID: 6HCR). Components of the MultiBac expression system can be obtained through material transfer agreements from I. P F , F G , I V B ; C, F G , F , R ; Em M W , C J W , G V , P F B ; P, I B , C V , J C , 10.1126/sciadv.aaw2853carried out negative-stain. C.S. C.S.31This work received generous support from the Oracle Higher Education and Research program to enable cryo-EM data processing using Oracle's high-performance public cloud infrastructure. B. Additional data related to this paper may be requested from the authors. SubmittedBB/L01386X/1). C.J.W. was supported by an EPSRC Research Software Engineering Fellowship (EP/N018591/1). C.S. acknowledges support from BBSRC (BB/P000940/1) and Wellcome Trust (210701/Z/18/Z). This work received generous support from the Oracle Higher Education and Research program to enable cryo-EM data processing using Oracle's high-performance public cloud infrastructure (https://cloud. oracle.com/en_US/cloud-infrastructure). Author contributions: P.F., F.G., and I.B. conceived the study. C.V., F.G., and F.R. expressed and purified all ADDomers with assistance from E.S. C.V. carried out most biochemical and biophysical work with input from P.F., assisted by E.S. J.C.B. determined and interpreted cryo-EM structures supervised by C.S. C.S. carried out negative-stain EM. M.W., C.J.W., G.V., and P.B. established cloud infrastructure, implemented cryo-EM software, and created workflows. C.T. and B.V. carried out initial mouse immunizations. V.J. and M.G. performed lymph node imaging experiments. L.C., together with C.V., prepared monocytes and dendritic cells for internalization experiments. P.F., I.B., C.V., and J.C.B. wrote the manuscript with input from all authors. Competing interests: P.F., F.G., and I.B. are inventors on a patent application related to this work filed by CNRS (no. PCT/ EP2017/057747, filed 31 March 2017). ADDomer is a registered trademark owned by Imophoron Ltd. The authors declare that they have no other competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Coordinates and EM data have been deposited at the public databases EMDB (EMD-0198) and PDB (PDB ID: 6HCR). Components of the MultiBac expression system can be obtained through material transfer agreements from I.B. Additional data related to this paper may be requested from the authors. Submitted 5 December 2018 Accepted 21 August 2019 Published 25 September 2019 10.1126/sciadv.aaw2853

Synthetic self-assembling ADDomer platform for highly efficient vaccination by genetically encoded multiepitope display. C Vragniau, J C Bufton, F Garzoni, E Stermann, F Rabi, C Terrat, M Guidetti, V Josserand, M Williams, C J Woods, G Viedma, P Bates, B Verrier, L Chaperot, C Schaffitzel, I Berger, P Fender, Sci. Adv. 52853Citation: C. Vragniau, J. C. Bufton, F. Garzoni, E. Stermann, F. Rabi, C. Terrat, M. Guidetti, V. Josserand, M. Williams, C. J. Woods, G. Viedma, P. Bates, B. Verrier, L. Chaperot, C. Schaffitzel, I. Berger, P. Fender, Synthetic self-assembling ADDomer platform for highly efficient vaccination by genetically encoded multiepitope display. Sci. Adv. 5, eaaw2853 (2019).


Aurelia: Jurnal Penelitian dan Pengabdian Masyarakat Indonesia Application of the Student Teams Achievement Division (STAD) Type Cooperative Learning Model to Improve Science Learning Outcomes of Class VIII Students at UPTD SMP Negeri 1 Alasa for the 2022/2023 Academic Year
July 2023

Erfin Noderius Hulu
Natalia K Lase
Aurelia: Jurnal Penelitian dan Pengabdian Masyarakat Indonesia Application of the Student Teams Achievement Division (STAD) Type Cooperative Learning Model to Improve Science Learning Outcomes of Class VIII Students at UPTD SMP Negeri 1 Alasa for the 2022/2023 Academic Year
July 2023Erfin Noderius Hulu & Natalia K. Lase -Universitas Nias 996Biology Education Study ProgramFaculty of Teacher Training and EducationUniversitas NiasGunungsitoli CityNorth Sumatera ProvinceIndonesia 12 Keywords: Student Teams Achievement Division and Learning Outcomes
Based on the results of initial observations at SMP Negeri 1 Alasa, several problems were found, namely: 1) Students were not actively involved in learning activities, and 2) Student learning outcomes in science subjects were still low. Research objectives: (1) Describe the implementation of the learning process through the application of cooperative learning models of the Student Teams Achievement Division type. (2) Describe student learning outcomes through the application of cooperative learning models of the Student Teams Achievement Division type. The research location is at SMP Negeri 1 Alasa. The research subjects were 30 students in class VIII-4, consisting of 16 boys and 14 girls. The research method used was classroom action research which consisted of four stages: (1) Planning, (2) Action, (3) Observation, and (4) Reflection. Research instruments: (1) observation sheet, (2) learning quality questionnaire, (3) student learning achievement test, (4) interview guide sheets, and (5) photo documentation. The results of the study: (1) Implementation of the learning process through the application of the Student Teams Achievement Division cooperative learning model in science subjects in Cycle I, namely 45.14% with poor criteria and Cycle II, namely 81.25% with good criteria. (2) Student learning outcomes through the application of the cooperative learning model type Student Teams Achievement Division in science subjects in Cycle I, namely 68.33 with sufficient criteria and Cycle II, namely 80.67 with good criteria.

INTRODUCTION

Education is a process in life that is very important in creating reliable and professional human resources. Education has a very decisive role as a vehicle for humanizing humans as a whole. Through education an intelligent, independent and creative generation can develop, as well as the formation of a dignified national character and civilization. For this reason, education is a joint responsibility of the government, society, as well as schools and teachers. In general, education takes place in schools in the form of teaching and learning activities between teachers and students. In the learning process the teacher is a very important component, because the success of the educational process is very dependent on the learning process designed and carried out by the teacher. Students learn a lot is determined by how the teacher implements learning. The teacher's task in this case is to manage learning actively and creatively so that learning causes changes in students both in cognitive, affective and psychomotor aspects as a result of students' responses to the stimulus received. With regard to these duties and responsibilities, teachers are expected to be able to choose appropriate learning strategies or methods so that they can actively involve students in learning to achieve predetermined competencies.

The 2013 curriculum is the curriculum that applies in the Indonesian education system. This curriculum is a curriculum implemented by the government to replace the 2006 curriculum, in which students are more active in the learning process and teachers use learning models that are appropriate to the material to be taught. Thus, the 2013 Curriculum is designed with the aim of preparing students to have the ability to live as individuals and citizens who are faithful, productive, creative, innovative and effective, and able to contribute to the life of society, nation and state.

Science learning basically has specific scientific characteristics that are different from other sciences. In accordance with the nature of science learning which refers to processes, products and scientific attitudes, science learning should ideally be able to provide a variety of learning experiences to understand science concepts and processes. These process skills include observing skills, submitting hypotheses, using tools and materials properly and correctly by always considering work safety and security, asking questions, classifying and interpreting data, and communicating findings orally or in writing, digging and sorting out relevant factual information. to test ideas or solve everyday problems.

Based on the results of a preliminary study conducted by prospective researchers at UPTD SMP Negeri 1 Alasa on January 24 2022 found several problems during the learning process activities. When learning activities take place students are less actively involved in learning activities. The delivery of subject matter to students is still dominant using conventional learning models, which means that learning is only teacher-centered. Students are less courageous in expressing opinions/questions. Students look less prepared in following the learning process. During the learning process activities the teacher rarely uses learning media, then the facilities and infrastructure needed to support the implementation of learning process activities are still limited. Based on the problems described above, the best solution in overcoming some of these problems is that the teacher must be able to find the best way to increase student involvement in learning process activities. One solution is to apply a cooperative learning model of the Student Teams Achievement Division (STAD) type.

The STAD type cooperative learning model is a learning model that is useful for fostering the ability to cooperate, be creative, think critically and have the ability to help friends and is a very simple cooperative learning. According to Nurhadi, et al., in Ade and Septi (2017: 50) put forward the advantages of the STAD type cooperative learning model, namely: (1) Students are more able to listen, accept, and respect others, (2) Students are able to identify their feelings as well as other people's feelings. others, (3) Students can receive experience and be understood by others, (4) Students are able to convince themselves for others, (5) Students are able to develop individual potential. In accordance with the description above, it can be concluded that through the application of the cooperative learning model type Student Teams Achievement Division (STAD) it is hoped that it will be able to train and develop students' abilities to learn, so that through this model it will be able to stimulate students' thinking skills to be good. The objectives in carrying out this research were: To describe the implementation of the learning process through the application of the Student Teams Achievement Division (STAD) cooperative learning model in science subjects. Describe student learning outcomes through the application of the cooperative learning model of the Student Teams Achievement Division (STAD) type in science subjects.

RESEARCH METHODS

In carrying out this research, researchers used the Classroom Action Research (PTK) method, so that the objects of action were: the application of the cooperative learning model of the Student Teams Achievement Divisions (STAD) type and student learning outcomes in science subjects. The research location was UPTD SMP Negeri 1 Alasa, Alasa District, North Nias Regency, North Sumatra Province. The research subjects were class VIII-4 students with a total of 30 people. This research was carried out in the odd semester of the 2022/2023 school year and adjusted to the schedule of science subjects. The duration of the research was approximately 1 month and each cycle held 3 meetings and one additional meeting for the end of the cycle. To collect data in this study used several research instruments. The research instruments used are as follows: 1. Observation sheet. Observation sheets are used to observe the learning process in class. This observation sheet was filled in by the science subject teacher as an observer. The observation sheets used by researchers include: Observation Sheets for the Learning Process (Teacher Respondents) and these observation sheets are used to collect data on the implementation of learning process activities carried out by researchers in the classroom. Student Activity Observation Sheet, Student activity observation sheet is a sheet that contains guidelines for carrying out observations of student activities during the learning process activities taking place in the classroom. Student Observation Sheets Who Are Not Actively Involved, This observation sheet is used to collect data about students who are not actively involved in the learning process. The grid or indicators in carrying out observations of students who are not actively involved are according to Isnawati, (2019: 5), namely: (1) noisy, (2) doing other assignments, (3) sleepy, (4) going in and out of class, ( 5) disturbing other students, (6) daydreaming, (7) being nosy, (8) scribbling on paper, (9) playing cellphones/games, and (10) moving seats. Based on the grid or indicators above, the researcher will use these indicators in carrying out observations of students who are not actively involved in the learning process activities. 2. Student Learning Outcomes Test. Learning outcomes test is used to determine the increase in student learning outcomes. Before the learning outcomes test is used as a research instrument, validation is carried out first and the instrument is tested. 3. Learning Quality Questionnaire Sheet. The learning quality questionnaire is an instrument for measuring the quality of learning which is arranged in the form of an objective questionnaire, where the respondents, in this case, are students who will be given a number of questions. Learning quality questionnaires were distributed to students (respondents) at the end of each cycle.

Research Design

In the implementation of this research will consist of 4 (four) stages, namely: planning, implementation, observation and reflection. Of the four stages, the actions in their implementation are as follows: 1. Planning (Planning), For each meeting, the researcher prepares: Prepares learning tools such as syllabus and lesson plans. Prepare learning materials and determine the topics of discussion in the learning process activities. Prepare the learning media that will be needed. Prepare observation sheets (observations), which consist of: Observation sheets of the learning process (teacher respondents). Student activity observation sheet. Observation sheets of students who are not actively involved. At the end of each cycle, the researcher prepares: Learning achievement tests, Learning quality questionnaires, Documentation (Photos). 2. Implementation (Action). The implementation of this research began in September s.d.

October 2022. Researchers carry out learning activities by applying the cooperative learning model of the Student Team Achievement Divisions (STAD) type in accordance with the plans that have been prepared by the researchers. Implementation of cycle I consisted of 3 meetings plus 1 meeting at the end of the cycle. In each meeting, learning was carried out using the cooperative learning model of the Student Team Achievement Divisions (STAD) type, while cycle II was carried out if the reflection results of cycle I did not reach the predetermined targets. Then in cycle II, 3 meetings were held plus 1 meeting at the end of the cycle. If the reflection results of cycle II have reached the target, the problem is resolved and research findings are formulated. If the reflection results of cycle II are not achieved, the problem has not been resolved and it is continued in the next cycle. 3. Observation (Observation). During the learning process, the science subject teacher acts as an observer by observing/assessing the suitability of the steps for implementing the cooperative learning model of the Student Team Achievement Divisions (STAD) type and paying attention to student activity during the learning process activities using observation sheets, while conducting interviews and giving the questionnaire will be carried out by the researcher at the end of each cycle. 4. Reflection (Reflection). Reflection is done at the end of each meeting and at the end of each cycle. Reflecting on the results or processing the results of observations made by observers concerning research instruments consisting of: At the end of each meeting, researchers as teachers recapitulate the results of observations of research instruments consisting of: Observation sheets of the learning process (teacher respondents), Observation sheets of student activities, Observation sheets of students who are not actively involved. At the end of each cycle, the researcher as the teacher recapitulates the results of observations of the research instruments which consist of: Learning achievement tests. Learning quality questionnaire. Documentation.

Action Implementation

In accordance with what was stated from the beginning, that this research was conducted in 2 cycles, with the following description: 1. Cycle I (First). The first cycle consists of 3 meetings plus 1 meeting for the end of the cycle.

In each meeting, learning was carried out using the cooperative learning model of the Student Team Achievement Divisions (STAD) type, where the steps of the learning process are listed in the lesson plan. During cycle I, the subject teacher as an observer will fill out the observation sheet according to the learning steps taken while the researcher acts as a teacher. After cycle I ended, an evaluation was then carried out by giving a learning achievement test to find out whether the learning outcomes had reached the KKM, and then holding a questionnaire and conducting interviews. Based on the reflection results of cycle I, the deficiencies or weaknesses in the reflection results of cycle I will be perfected in cycle II. 2. Cycle II (Second). After evaluating the results of the implementation of the first cycle, if it still does not achieve the expected results, then proceed to the second cycle. In the implementation of the second cycle consists of 3 meetings plus 1 meeting at the end of the cycle to administer the learning outcomes test. The action in cycle II is to perfect the deficiencies in the previous meeting.

RESEARCH RESULTS AND DISCUSSION

This research was conducted at Alasa 1 Public Middle School, located in Ombolata Village, Alasa District, North Nias Regency. The research subjects were 30 students in class VIII-4, consisting of 16 boys and 14 girls. Before the research was carried out, the researcher first collaborated with the Principal of SMP Negeri 1 Alasa and with his approval the research could be carried out and the researcher collaborated with the science subject teacher. The implementation of this research includes four stages: planning, action, observation, and reflection. The research was carried out using the services of an observer or observer, namely a science subject teacher who assisted in carrying out observations during the research, so that this research activity could be carried out properly. Research activities are carried out to coincide with science class hours and do not interfere with the implementation of other learning processes and researchers as well as practitioners do not need to leave the classroom where they teach. This research begins on October 10 to November 21, 2022. Classroom action research on science learning will apply a cooperative learning model of the Student Teams Achievement Division (STAD) type which aims to improve the quality of learning and student learning outcomes in science subjects.

Cycle I 1. The first meeting, Cycle I. The implementation of the learning process activities at this meeting is still lacking, this is in accordance with the results of observations of the learning process (teacher respondents) obtained an average observation result of 1.42 and the percentage of observations is 35.42%. . Then the results of observations of students who were actively involved in the learning process obtained the percentage of 55.42%, this result showed that students were not fully involved in participating in the learning process activities. In the observation results section of students who are not actively involved in the learning process, the percentage of observations is 20%. At this meeting there were 6 students who were not actively involved, namely there were students who were noisy, sleepy, going in and out of class, and changing seats. The solution in overcoming this problem is that researchers will advise students who are not active during the learning process activities. 2. Second Meeting, Cycle I. At this meeting the implementation of learning activities has not been carried out optimally. In accordance with the results of observations of the learning process (teacher respondents) the average observation result was 1.75 and the percentage of observations was 43.75%. Then the results of observations of students who were actively involved in the learning process activities, the percentage of which was 64.17% was classified as sufficient criteria. The results of observations of students who were not actively involved in the learning process activities obtained the percentage of observations, namely 16.67%. At this meeting there were 5 students who were less actively involved, namely there were students who were noisy, doing other assignments, and there were students who were sleepy. 3. Third Meeting, Cycle I. At this meeting the implementation of the learning process activities underwent changes compared to the previous meeting. In accordance with the results of observations of the learning process (teacher respondents) the average observation result was 2.25 and the percentage of observations was 56.25%. Then on the results of observations of students who are actively involved in the learning process activities the percentage is 68.75%. These results indicate that there has been an increase in students who are actively involved in learning activities. Furthermore, the results of observations of students who were not actively involved in the learning process obtained the percentage of observations, namely 13.33%. At this meeting there were 4 students who were less actively involved, namely there were students who were noisy, going in and out of class, there were students daydreaming, and disturbing other students. 4. End of Cycle I. At the end of Cycle I, the percentage of the learning quality questionnaire was obtained, namely 64.09%, which was classified as sufficient criteria. Then the percentage of completeness of student learning outcomes is 56.67% while the percentage of students who do not complete is 43.33%, and the average student learning outcomes is 68.33 belonging to the sufficient criterion. According to the recapitulation results, the average reflection result at the end of Cycle I was 57.17%. These results indicate that the implementation of Cycle I still has not met expectations, which means that the problems in Cycle I have not been resolved, so that it will continue in Cycle II. Some of the weaknesses found in the implementation of Cycle I were due to several things, which included: 1. The teacher's ability to master learning material is still lacking. 2. The ability to organize learning tasks in the application of the cooperative learning model of the Student Teams Achievement Division type is still lacking in every activity of the learning process. 3. The teacher's ability to control the class is still lacking. 4. Researchers still lack mastery over all stages of implementing the cooperative learning model of the Student Teams Achievement Division type. 5. The ability to solve problems in the application of the cooperative learning model type Student Teams Achievement Division is still lacking. 6. The ability to provide feedback and evaluate learning activities is still not optimal.

Based on some of the weaknesses above, the researchers took corrective actions which included: 1. Prepare well in mastering the subject matter to be taught. 2. Prepare yourself well in mastering the stages of implementing the cooperative learning model of the Student Teams Achievement Division type. 3. Prepare yourself well in organizing learning tasks in the application of the STAD type cooperative learning model. 4. Always motivate students to be more actively involved in participating in the learning process. 5. Prepare yourself well in mastering the class and in using learning media. 6. Improving the ability to provide feedback and evaluate learning activities optimally.

Cycle II

1. First Meeting, Cycle II. At this meeting the implementation of learning has experienced a good improvement compared to the previous meeting. In accordance with the results of observations of learning process activities (teacher respondents) the average observation result was 2.92 and the percentage of observations was 72.92%. Then the results of observations of students who are actively involved in the learning process activities are 75.42%. Based on these results it can be concluded that some students have begun to be able to be actively involved in participating in the learning process. Furthermore, the results of observations of students who were not actively involved in the learning process obtained the percentage of observations, namely 13.33%. At this meeting there were still 4 students who were not actively involved, namely there were students who were noisy and doing other assignments. 2. Second Meeting, Cycle II. At this meeting the implementation of learning has increased compared to the previous meeting. In accordance with the results of observations of the learning process (teacher respondents) the average observation result was 3.17 and the percentage of observations was 79.17%. Then the results of observations of students who were actively involved in the learning process activities were 80.83%. Based on these results it can be concluded that there is an increase in students who are actively involved in participating in the learning process. Then on the results of observations of students who were not actively involved in the learning process, the percentage of observations was obtained, namely 10%. At this meeting there were still 3 students who were not actively involved, namely there were students who were noisy, sleepy, and in and out of class. 3. Third Meeting, Cycle II. At this meeting the implementation of learning has increased compared to the previous meeting. In accordance with the results of observations of the learning process (teacher respondents) the average observation result was 3.67 and the percentage of observations was 91.67%. As for the results of observations of students who were actively involved in the learning process activities, namely 88.75%. Based on these results it can be concluded that there is an increase in students who are actively involved in participating in the learning process. Then the results of observations of students who were not actively involved in the learning process obtained the percentage of observations, namely 6.67%. At this meeting there were still 2 students who were not actively involved, namely there were students who daydreamed and disturbed other students. 4. End of Cycle II. At the end of Cycle II, the percentage of the learning quality questionnaire was obtained, namely 95.44%, classified as very good criteria (Appendix 33.c). Then the percentage of completeness of student learning outcomes is 86.67% while the percentage of students who do not complete is 13.33% and the average student learning outcomes is 80.67 belonging to good criteria. According to the recapitulation results, the average reflection result for the end of Cycle II was 86.26%. These results indicate that the implementation of research in Cycle II has been achieved and has met expectations, and in the results of interviews stated that students are happy and interested in participating in learning activities through the application of the Student Teams Achievement Division type cooperative learning model because through the application of the Student Teams Achievement Division learning model students can be actively involved in learning activities so that student learning outcomes are good and the quality of learning is good. So, it can be concluded that the implementation of Cycle II has met expectations so that this research has been achieved optimally.

CONCLUSION

Based on the processing and analysis of research data that has been carried out, the researchers conclude as follows: Implementation of the learning process through the application of cooperative learning models of the Student Teams Achievement Division type in science subjects in Cycle I, namely 45.14% with less criteria and Cycle II, namely 81 .25% with good criteria. Student learning outcomes through the application of the cooperative learning model type Student Teams Achievement Division in science subjects in Cycle I, namely 68.33 with sufficient criteria and Cycle II, namely 80.67 with good criteria. Based on the research results, the author's suggestions are: Teachers should use the Student Teams Achievement Division cooperative learning model because it is able to develop students' attitudes and skills in critical thinking and mutual respect for each other. A teacher who wants to apply the Student Teams Achievement Division cooperative learning model must fully master the implementation stage in order to obtain optimal results.

BIBLIOGRAPHY

Ade, Nurlatifah dan Septi Ambarwati. 2017. Pengaruh Model Pembelajaran Kooperatif Tipe Student Teams Achievement Division (STAD) Terhadap Hasil Belajar IPA Ditinjau Dari Keaktifan Siswa. Vol 4. No 2. Jurnal Ilmiah Pendidikan IPA. (online). (https://jurnal. ustjogja.ac.id/index.php/ NATURAL/article/view/1854/1012 diakses pada 30 Maret 2022)Aurelia: Jurnal Penelitian dan Pengabdian Masyarakat Indonesia
E-ISSN: 2964-2493 P-ISSN: 2962-0430
Vol. 2 No. 2 July 2023

Nomor 2. (Online). Nomor 2. (Online).

Pelatihan dan Pendampingan Penyusunan Proposal Penelitian Tindakan. Dkk Budasi, Kelas Bagi Guru-Guru SMA/SMK/Madrasah Se-Kabupaten Bima-NTBProsiding SENADIMAS Ke-4. (onlineBudasi, dkk. 2019. Pelatihan dan Pendampingan Penyusunan Proposal Penelitian Tindakan Kelas Bagi Guru-Guru SMA/SMK/Madrasah Se-Kabupaten Bima-NTB. Prosiding SENADIMAS Ke-4. (online).

Penggunaan Model Pembelajaran Kooperatif Tipe Jigsaw Terhadap Kemampuan Pemahaman Konsep Belajar Siswa. Dkk Darmawan, 8Nomor 1. (onlineDarmawan, dkk. 2022. Penggunaan Model Pembelajaran Kooperatif Tipe Jigsaw Terhadap Kemampuan Pemahaman Konsep Belajar Siswa. Volume 8 Nomor 1. (online). (http://ejurnal.pps.ung.ac.id/index.php/ Aksara/article/view/835/686 diakses pada 16 Mei 2022)

Pengaruh Model Pembelajaran Kooperatif Tipe Ttw Untuk Menulis Karangan Persuasi. Damanik Elviana, Lili Dan, Tansliova, 2Nomor 1. (onlineElviana, Damanik dan Lili Tansliova. 2020. Pengaruh Model Pembelajaran Kooperatif Tipe Ttw Untuk Menulis Karangan Persuasi. Volume 2 Nomor 1. (online). (http://jurnal.usi.ac.id/index.php/artikulasi/article/ view/150/137 diakses pada 16 Mei 2022)

Meningkatkan Hasil Belajar Fisika Melalui Penggunaan Metode Demonstrasi Dan Media Audiovisual Pada Siswa Kelas X MAN 4 Jakarta. Andragogi Jurnal Diklat Teknis. Volume VI. Nomor 2. (online). Hernawati Eneng, Eneng, Hernawati. 2018. Meningkatkan Hasil Belajar Fisika Melalui Penggunaan Metode Demonstrasi Dan Media Audiovisual Pada Siswa Kelas X MAN 4 Jakarta. Andragogi Jurnal Diklat Teknis. Volume VI. Nomor 2. (online). (https://pusdiklattekniskemenag.e- journal.id/andragogi/article/ view/60/52 diakses pada 1 April 2022)

Penerapan Model Pembelajaran Kooperatif Tipe Student Teams Achievement Division Terhadap Motivasi Belajar Peserta Didik Di SMAN 8 Banda Aceh. Rini Ernawita Dan, Safitri, Jurnal Pendidikan Sains Indonesia. 06No.01. (onlineErnawita dan Rini Safitri. 2018. Penerapan Model Pembelajaran Kooperatif Tipe Student Teams Achievement Division Terhadap Motivasi Belajar Peserta Didik Di SMAN 8 Banda Aceh. Jurnal Pendidikan Sains Indonesia. Vol. 06. No.01. (online). (http://e- repository.unsyiah.ac.id/JPSI/article/ download/10713/8877 diakses pada 24 Maret 2022)

Hakikat Pembelajaran Yang Efektif. Jurnal At-Tafkir. Volume XI. Nomor 1. (online). Fakhrurrazi, Fakhrurrazi. 2018. Hakikat Pembelajaran Yang Efektif. Jurnal At-Tafkir. Volume XI. Nomor 1. (online). (https://journal.iainlangsa.ac.id/index.php/at/ article/view/529/331 diakses pada 2 April 2022)

Penerapan Metode Giving Question and Getting Answer untuk Meningkatkan Hasil Belajar Peserta Didik pada Mata Pelajaran Pendidikan Agama Islam. Yulianti Hesti, Jurnal Penelitian Pendidikan Islam. 6Nomor 1. (onlineHesti, Yulianti, dkk. 2018. Penerapan Metode Giving Question and Getting Answer untuk Meningkatkan Hasil Belajar Peserta Didik pada Mata Pelajaran Pendidikan Agama Islam. Jurnal Penelitian Pendidikan Islam. Volume 6. Nomor 1. (online). (https://riset- iaid.net/index.php/jppi/ article/view/297/348 diakses pada 6 April 2022)

Model-Model Pengajaran dan Pembelajaran Isu-Isu Metodis dan Paradigmatis. Miftahul Huda, Pustaka PelajarYogyakartaHuda, Miftahul. 2017. Model-Model Pengajaran dan Pembelajaran Isu-Isu Metodis dan Paradigmatis. Yogyakarta: Pustaka Pelajar.

Penerapan Model Pembelajaran Student Teams Achievement Divisions (STAD) dalam Meningkatkan Hasil Belajar IPA Peserta Didik SMP. Andi Rofina, Andi Dan, Rugaiyah, Journal of Islamic Education and Teacher Training. 21Rofina, Andi dan Andi Rugaiyah. 2020. Penerapan Model Pembelajaran Student Teams Achievement Divisions (STAD) dalam Meningkatkan Hasil Belajar IPA Peserta Didik SMP. Journal of Islamic Education and Teacher Training. Vol. 2. No.1. (online). (http://jurnal. mtsddicilellang. sch.id/index.php/al-musannif/article/view/40/20 diakses pada 30 Maret 2022)

Peningkatan Kualitas Pembelajaran Geometri Berbasis Discovery Learning Melalui Model Think Pair Share. Ayu Shindia, Dkk Rega Puspita, Joyful Learning Journal. 2Nomor 3. (online)Shindia, Ayu Rega Puspita, dkk. 2017. Peningkatan Kualitas Pembelajaran Geometri Berbasis Discovery Learning Melalui Model Think Pair Share. Joyful Learning Journal. Volume 2. Nomor 3. (online). (https:// journal.unnes.ac.id/sju/index.php/jlj/article/view/2058/1874 diakses pada 1 April 2022)

Statistika Untuk Penelitian. Bandung: Alfabeta Sugiyono. 2019. Metode Penelitian Kuantitatif Kualitatif dan R & D. Bandung: Alfabeta Sustin, Sumarni Burengge. 2020. Penerapan Model Pembelajaran Kooperatif Tipe STAD dengan Pendekatan Kontekstual bagi Siswa SDN 7 Tentena Sulawesi Tengah. Jurnal Paedagogy. 7Nomor 4. (onlineSugiyono. 2017. Statistika Untuk Penelitian. Bandung: Alfabeta Sugiyono. 2019. Metode Penelitian Kuantitatif Kualitatif dan R & D. Bandung: Alfabeta Sustin, Sumarni Burengge. 2020. Penerapan Model Pembelajaran Kooperatif Tipe STAD dengan Pendekatan Kontekstual bagi Siswa SDN 7 Tentena Sulawesi Tengah. Jurnal Paedagogy. Volume 7. Nomor 4. (online). (http://e- journal.undikma.ac.id/index.php/pedagogy/article/download/ 2832/2002 diakses pada 30 Maret 2022)

Pengembangan Media Pembelajaran Untuk Meningkatkan Hasil Belajar Siswa. Nurrita Teni, 03Nomor 01. (onlineTeni, Nurrita. 2018. Pengembangan Media Pembelajaran Untuk Meningkatkan Hasil Belajar Siswa. Volume 03. Nomor 01. (online).

. Dkk Vivi, Meningkatkan Kemampuan Mengklasifikasi Melalui Bermain Konstruktif Sifat Padat. 03Nomor 02. (onlineVivi, dkk. 2018. Meningkatkan Kemampuan Mengklasifikasi Melalui Bermain Konstruktif Sifat Padat. Volume 03. Nomor 02. (online).

Dkk Widoyoko, Strategi Belajar Mengajar. Jakarta: PT. Raja Grafindo Persada. Zainal, Aqib dan M. Chotibuddin. YogyakartaDeepublishTeori dan Aplikasi Penelitian Tindakan KelasWidoyoko, dkk. 2018. Strategi Belajar Mengajar. Jakarta: PT. Raja Grafindo Persada. Zainal, Aqib dan M. Chotibuddin. 2018. Teori dan Aplikasi Penelitian Tindakan Kelas. Yogyakarta: Deepublish.