Optimization and Validation of a Liquid Formulation for a New Recombinant Veterinary Product using QbD Approach.

Protein formulation and drug characterization are one of the most difficult and time-consuming tasks because of the complexity of biotherapeutic proteins. Hence, maintaining a protein drug in its active state typically requires preventing changes in its physical and chemical properties. Quality by Design (QbD) is a systematic approach emphasizing product and process understanding. Design of Experiments (DoE) is one of the most important QbD tools, allowing the possibility to modify the formulation attributes within a defined design space. Here, we report the validation of a RP-HPLC assay for recombinant equine chorionic gonadotropin (reCG) that demonstrated a high correlation with the in vivo potency biological assay. QbD concepts were then applied to obtain an optimized liquid formulation of reCG with a predefined quality product profile. The developed strategy demonstrates the importance of applying multivariable strategies as DoE to simplify formulation stages, improving the quality of the obtained results. Moreover, it is important to highlight that this is the first time that a liquid formulation is reported for an eCG molecule, since, up to now, the only eCG products available in the market for veterinary use consisted in partially purified preparations of pregnant mare serum gonadotropin (PMSG) presented as a lyophilized product.

Design and characterization of chimeric Rabies-SARS-CoV-2 virus-like particles for vaccine purposes.

Due to the high number of doses required to achieve adequate coverage in the context of COVID-19 pandemics, there is a great need for novel vaccine developments. In this field, there have been research approaches that focused on the production of SARS-CoV-2 virus-like particles. These are promising vaccine candidates as their structure is similar to that of native virions but they lack the genome, constituting a biosafe alternative. In order to produce these structures using mammal cells, it has been established that all four structural proteins must be expressed. Here we report the generation and characterization of a novel chimeric virus-like particle (VLP) that can be produced by the expression of a single novel fusion protein that contains SARS-CoV-2 spike (S) ectodomain fused to rabies glycoprotein membrane anchoring region in HEK293 cells. This protein is structurally similar to native S and can autonomously bud forming enveloped VLPs that resemble native virions both in size and in morphology, displaying S ectodomain and receptor binding domain (RBD) on their surface. As a proof of concept, we analyzed the immunogenicity of this vaccine candidate in mice and confirmed the generation of anti-S, anti-RBD, and neutralizing antibodies. KEY POINTS: • A novel fusion rabies glycoprotein containing S ectodomain was designed. • Fusion protein formed cVLPs that were morphologically similar to SARS-CoV-2 virions. • cVLPs induced anti-S, anti-RBD, and neutralizing antibodies in mice.

A COVID-19 vaccine candidate based on SARS-CoV-2 spike protein and immune-stimulating complexes.

Spike protein from SARS-CoV-2, the etiologic agent of the COVID-19 pandemic disease, constitutes a structural protein that proved to be the main responsible for neutralizing antibody production. Thus, its sequence is highly considered for the design of candidate vaccines. Animal cell culture represents the best option for the production of subunit vaccines based on recombinant proteins since they introduce post-translational modifications that are important to mimic the natural antigenic epitopes. Particularly, the human cell line HEK293T has been explored and used for the production of biotherapeutics since the products derived from them present human-like post-translational modifications that are important for the protein's activity and immunogenicity. The aim of this study was to produce and characterize a potential vaccine for COVID-19 based on the spike ectodomain (S-ED) of SARS-CoV-2 and two different adjuvants: aluminum hydroxide (AH) and immune-stimulating complexes (ISCOMs). The S-ED was produced in sHEK293T cells using a 1-L stirred tank bioreactor operated in perfusion mode and purified. S-ED characterization revealed the expected size and morphology. High N-glycan content was confirmed. S-ED-specific binding with the hACE2 (human angiotensin-converting enzyme 2) receptor was verified. The immunogenicity of S-ED was evaluated using AH and ISCOMs. Both formulations demonstrated the presence of anti-RBD antibodies in the plasma of immunized mice, being significantly higher for the latter adjuvant. Also, higher levels of IFN-γ and IL-4 were detected after the ex vivo immune stimulation of spleen-derived MNCs from ISCOMs immunized mice. Further analysis confirmed that S-ED/ISCOMs elicit neutralizing antibodies against SARS-CoV-2. KEY POINTS: Trimeric SARS-CoV-2 S-ED was produced in stable recombinant sHEK cells in serum-free medium. A novel S-ED vaccine formulation induced potent humoral and cellular immunity. S-ED formulated with ISCOMs adjuvant elicited a highly neutralizing antibody titer.

Physicochemical Characterization of a Recombinant eCG and Comparative Studies with PMSG Commercial Preparations.

Equine chorionic gonadotropin (eCG) is a glycoprotein hormone widely used in timed artificial ovulation (TAI) and superovulation protocols to improve the reproductive performance in livestock. Until recently, the only eCG products available in the market for veterinary use consisted in partially purified preparations of pregnant mare serum gonadotropin (PMSG). Here, a bioactive recombinant eCG (reCG) produced in suspension CHO-K1 cells was purified employing different chromatographic methods (hydrophobic interaction chromatography and reverse-phase (RP)-HPLC) and compared with a RP-HPLC-purified PMSG. To gain insight into the structural and functional characteristics of reCG, a bioinformatics analysis was performed. An exhaustive characterization comprising the determination of the purity degree, aggregates and nicked forms through SDS-PAGE, RP-HPLC and SEC-HPLC was performed. Higher order structures were studied by fluorescence spectroscopy and SEC-HPLC. Isoforms profile were analyzed by isoelectric focusing. Glycosylation analysis was performed through pulsed amperometric detection and PNGase F treatment following SDS-PAGE and weak anion exchange-HPLC. Slight differences between the purified recombinant hormones were found. However, recombinant molecules and PMSG exhibited variations in the glycosylation pattern. In fact, differences in sialic acid content between two commercial preparations of PMSG were also obtained, which could lead to differences in their biological potency. These results show the importance of having a standardized production process, as occurs in a recombinant protein bioprocess. Besides, our results reflect the importance of the glycan moieties on eCG conformation and hence in its biological activity, preventing denaturing processes such as aggregation.

The Overweight Paradox: Impact of Body Mass Index on Patients Undergoing VATS Lobectomy or Segmentectomy.

The aim of this study was to assess the impact of BMI on perioperative outcomes in patients undergoing VATS lobectomy or segmentectomy. Data from 5088 patients undergoing VATS lobectomy or segmentectomy, included in the VATS Group Italian Registry, were collected. BMI (kg/m2) was categorized according to the WHO classes: underweight, normal, overweight, obese. The effects of BMI on outcomes (complications, 30-days mortality, DFS and OS) were evaluated with a linear regression model, and with a logistic regression model for binary endpoints. In overweight and obese patients, operative time increased with BMI value. Operating room time increased by 5.54 minutes (S.E. = 1.57) in overweight patients, and 33.12 minutes (S.E. = 10.26) in obese patients (P < 0.001). Compared to the other BMI classes, overweight patients were at the lowest risk of pulmonary, acute cardiac, surgical, major, and overall postoperative complications. In the overweight range, a BMI increase from 25 to 29.9 did not significantly affect the length of stay, nor the risk of any complications, except for renal complications (OR: 1.55; 95% CI: 1.07-2.24; P = 0.03), and it reduced the risk of prolonged air leak (OR: 0.8; 95% CI: 0.71-0.90; P < 0.001). 30-days mortality is higher in the underweight group compared to the others. We did not find any significant difference in DFS and OS. According to our results, obesity increases operating room time for VATS major lung resection. Overweight patients are at the lowest risk of pulmonary, acute cardiac, surgical, major, and overall postoperative complications following VATS resections. The risk of most postoperative complications progressively increases as the BMI deviates from the point at the lowest risk, towards both extremes of BMI values. Thirty days mortality is higher in the underweight group, with no differences in DFS and OS.

Design and optimization of an IgG human ELISA assay reactive to recombinant RBD SARS-CoV-2 protein.

Serology assays are essential tools to mitigate the effect of COVID-19, help to identify previous SARS-CoV-2 infections or vaccination, and provide data for surveillance and epidemiologic studies. In this study, we report the production and purification process of the receptor-binding domain (RBD) of SARS-CoV-2 in HEK293 cells, which allowed the design, optimization, and validation of an indirect ELISA (iELISA) for the detection of human anti-RBD antibodies. To find the optimal conditions of this iELISA, a multivariate strategy was performed throughout design of experiments (DoE) and response surface methodology (RSM), one of the main tools of quality by design (QbD) approach. The adoption of this strategy helped to reduce the time and cost during the method development stage and to define an optimum condition within the analyzed design region. The assay was then validated, exhibiting a sensitivity of 94.24 (86.01-98.42%; 95% CI) and a specificity of 95.96% (89.98-98.89%; 95% CI). Besides, the degree of agreement between quality results assessed using kappa's value was 0.92. Hence, this iELISA represents a high-throughput technique, simple to perform, reliable, and feasible to be scaled up to satisfy the current demands. Since RBD is proposed as the coating antigen, the intended use of this iELISA is not only the detection of previous exposure to the virus, but also the possibility of detecting protective immunity. KEY POINTS: • RBD was produced in 1-L bioreactor and highly purified. • An iELISA assay was optimized applying QbD concepts. • The validation procedure demonstrated that this iELISA is accurate and precise.

Development, Production, and Characterization of Hepatitis B Subviral Envelope Particles as a Third-Generation Vaccine.

Vaccination still represents the most efficient and inexpensive strategy in the control of hepatitis B virus (HBV) infection. However, about 10% of the population vaccinated with the current S yeast-derived vaccine fail to induce an adequate immune response. Our group has developed a new-generation hepatitis B vaccine candidate composed by the three surface proteins of the HBV. Here we describe the methods to develop and characterize a stable CHO-K1 recombinant cell line able to produce and secrete hepatitis B subviral envelope particles (HBV-SVPs) containing L and M glycoproteins in addition to S glycoprotein. In addition, Western blot and immunogold electron microscopy techniques to evaluate the size, morphology, and composition of the particles are explained. Finally, immunization protocols are described in order to study the immunogenicity of HBV-SVPs and the ability of the antibodies triggered by these particles to recognize the binding site of HBV with the hepatocyte.

Rational design of novel fusion rabies glycoproteins displaying a major antigenic site of foot-and-mouth disease virus for vaccine applications.

Chimeric virus-like particles are self-assembling structures composed of viral proteins that had been modified to incorporate sequences from different organisms, being able to trigger immune responses against the heterologous sequence. However, the identification of suitable sites for that purpose in the carrier protein is not an easy task. In this work, we describe the generation of rabies chimeric VLPs that expose a major antigenic site of foot-and-mouth disease virus (FMDV) by identifying suitable regions in rabies glycoprotein (RVG), as a proof of concept of a novel heterologous display platform for vaccine applications. To identify adequate sites for insertion of heterologous sequences without altering the correct folding of RVG, we identified regions that were evolutionally non-conserved in Lyssavirus glycoproteins and performed a structural analysis of those regions using a 3D model of RVG trimer that we generated. The heterologous sequence was inserted in three different sites within RVG sequence. In every case, it did not affect the correct folding of the protein and was surface exposed, being recognized by anti-FMDV antibodies in expressing cells as well as in the surface of VLPs. This work sets the base for the development of a heterologous antigen display platform based on rabies VLPs. KEY POINTS: • Adequate regions for foreign epitope display in RVG were found. • G-H loop of FMDV was inserted in three regions of RVG. • The foreign epitope was detected by specific antibodies on fusion proteins. • G-H loop was detected on the surface of chimeric VLPs.

Detection and quantification of anti-rabies glycoprotein antibodies: current state and perspectives.

Rabies is an ancient fatal disease with no other available treatment than post-exposure vaccination, where the bite of infected animals, mainly dogs, is the leading cause of its transmission to human beings. In this context, global vaccination campaigns of companion animals, as well as wildlife reservoirs vaccination, are key factors to achieve the "Zero by 30" plan that pursues the eradication of dog-mediated human rabies by 2030. Rabies virus-neutralizing antibodies (VNAs) play an essential role in the disease protection, as it correlates with an adequate immune response and allows evaluating pre- or post-exposure prophylaxis efficacy. Hence, counting with reliable, accurate, and robust serological tests is of paramount importance. Currently, RFFIT and FAVN are the gold standard VNAs tests recommended by both the WHO and the OIE. Despite these methodologies are efficient and widely used, they present several drawbacks, as they are less easily to standardize and require the use of live rabies virus, containment facilities, and skilled professionals. Thus, in this review, we describe the state-of-the-art of alternative analytical methodologies currently available for rabies serology, with novel approaches based on pseudotyped recombinant viruses and emphasizing in the antigen binding methodologies that detect and quantify antibodies against the rabies glycoprotein. We discussed the wide range of assays that are interesting tools for a faster measurement of anti-rabies glycoprotein antibodies and, in some cases, less complex and more versatile than the gold standard methods. Finally, we discussed the key issues during the design and optimization steps of ELISA assays, highlighting the importance of validation and standardization procedures to improve rabies serology tests and, as a consequence, their results. KEY POINTS: • An exhaustive revision of rabies serology testing was made. • No rabies serology assay can be thought as better than others for all intents and purposes. • The validation procedure guarantees reliable and consistent results among the globe.

Development of a suitable manufacturing process for production of a bioactive recombinant equine chorionic gonadotropin (reCG) in CHO-K1 cells.

Equine chorionic gonadotropin (eCG) is a heterodimeric glycoprotein hormone produced by pregnant mares that has been used to improve reproductive performance in different domestic species. Several strategies to produce the hormone in a recombinant way have been reported; nevertheless, no approach has been able to produce a recombinant eCG (reCG) with significant in vivo bioactivity or in sufficient quantities for commercial purposes. For this reason, the only current product available on the market consists of partially purified preparations from serum of pregnant mares (PMSG). Herein, we describe a highly efficient process based on third-generation lentiviral vectors as delivery method for the production of reCG in suspension CHO-K1 cells, with productivities above 20 IU 106 cell-1.d-1 and 70% purification yields after one purification step. Importantly, reCG demonstrated biological activity in cattle, since around 30 µg of reCG were needed to exert the same biologic effect of 400 IU of PMSG in an ovulation synchronization protocol. The results obtained demonstrate that the developed strategy represents an attractive option for the production of reCG and constitutes an auspicious alternative for the replacement of animals as a source of PMSG.

Chimeric VLPs Based on HIV-1 Gag and a Fusion Rabies Glycoprotein Induce Specific Antibodies against Rabies and Foot-and-Mouth Disease Virus.

Foot and mouth disease is a livestock acute disease, causing economic losses in affected areas. Currently, control of this disease is performed by mandatory vaccination campaigns using inactivated viral vaccines. In this work, we describe the development of a chimeric VLP-based vaccine candidate for foot-and-mouth disease virus (FMDV), based on the co-expression of the HIV-1 Gag protein and a novel fusion rabies glycoprotein (RVG), which carries in its N-term the FMDV main antigen: the G-H loop. It is demonstrated by confocal microscopy that both Gag-GFP polyprotein and the G-H loop colocalize at the cell membrane and, that the Gag polyprotein of the HIV virus acts as a scaffold for enveloped VLPs that during the budding process acquires the proteins that are being expressed in the cell membrane. The obtained VLPs were spherical particles of 130 ± 40 nm in diameter (analyzed by TEM, Cryo-TEM and NTA) carrying an envelope membrane that efficiently display the GH-RVG on its surface (analyzed by gold immunolabeling). Immunostainings with a FMDV hyperimmune serum showed that the heterologous antigenic site, genetically fused to RVG, is recognized by specific G-H loop antibodies. Additionally, the cVLPs produced expose the G-H loop to the liquid surrounding (analyzed by specific ELISA). Finally, we confirmed that these FMD cVLPs are able to induce a specific humoral immune response, based on antibodies directed to the G-H loop in experimental animals.

Recommendations from the Italian intersociety consensus on Perioperative Anesthesia Care in Thoracic surgery (PACTS) part 1: preadmission and preoperative care.

INTRODUCTION: Anesthetic care in patients undergoing thoracic surgery presents specific challenges that necessitate standardized, multidisciplionary, and continuously updated guidelines for perioperative care. METHODS: A multidisciplinary expert group, the Perioperative Anesthesia in Thoracic Surgery (PACTS) group, comprising 24 members from 19 Italian centers, was established to develop recommendations for anesthesia practice in patients undergoing thoracic surgery (specifically lung resection for cancer). The project focused on preoperative patient assessment and preparation, intraoperative management (surgical and anesthesiologic care), and postoperative care and discharge. A series of clinical questions was developed, and PubMed and Embase literature searches were performed to inform discussions around these areas, leading to the development of 69 recommendations. The quality of evidence and strength of recommendations were graded using the United States Preventative Services Task Force criteria. RESULTS: Recommendations for preoperative care focus on risk assessment, patient preparation (prehabilitation), and the choice of procedure (open thoracotomy vs. video-assisted thoracic surgery). CONCLUSIONS: These recommendations should help pulmonologists to improve preoperative management in thoracic surgery patients. Further refinement of the recommendations can be anticipated as the literature continues to evolve.

Recommendations from the Italian intersociety consensus on Perioperative Anesthesa Care in Thoracic surgery (PACTS) part 2: intraoperative and postoperative care.

INTRODUCTION: Anesthetic care in patients undergoing thoracic surgery presents specific challenges that require a multidisciplinary approach to management. There remains a need for standardized, evidence-based, continuously updated guidelines for perioperative care in these patients. METHODS: A multidisciplinary expert group, the Perioperative Anesthesia in Thoracic Surgery (PACTS) group, was established to develop recommendations for anesthesia practice in patients undergoing elective lung resection for lung cancer. The project addressed three key areas: preoperative patient assessment and preparation, intraoperative management (surgical and anesthesiologic care), and postoperative care and discharge. A series of clinical questions was developed, and literature searches were performed to inform discussions around these areas, leading to the development of 69 recommendations. The quality of evidence and strength of recommendations were graded using the United States Preventive Services Task Force criteria. RESULTS: Recommendations for intraoperative care focus on airway management, and monitoring of vital signs, hemodynamics, blood gases, neuromuscular blockade, and depth of anesthesia. Recommendations for postoperative care focus on the provision of multimodal analgesia, intensive care unit (ICU) care, and specific measures such as chest drainage, mobilization, noninvasive ventilation, and atrial fibrillation prophylaxis. CONCLUSIONS: These recommendations should help clinicians to improve intraoperative and postoperative management, and thereby achieve better postoperative outcomes in thoracic surgery patients. Further refinement of the recommendations can be anticipated as the literature continues to evolve.

Characterization of hepatitis B virus surface antigen particles expressed in stably transformed mammalian cell lines containing the large, middle and small surface protein.

Vaccination still represents the most efficient and inexpensive strategy in the control of hepatitis B virus (HBV) infection. However, about 10% of the population vaccinated with the current yeast derived vaccine, consisting of the non-glycosylated form of the small envelope protein (S) of the HBV, fail to display an adequate immune response. Therefore, there is a need for the development of new vaccines with enhanced immunogenicity. On this regard, new generation vaccines containing L and preS2-containing HBV surface proteins in addition to S, have proven to be able to bypass the lack of response of the standard vaccine. In this work, we describe the development of stable recombinant CHO-K1 and HEK293 cell lines able to produce and secrete hepatitis B subviral envelope particles (HBV-SVPs) composed by the three surface proteins of the HBV. In turn, we demonstrated that these particles induced a specific humoral immune response in experimental animals and triggered the production of antibodies with the ability to recognize the binding site of HBV with the hepatocyte. Thus, these HBV-SVPs represent a promising candidate as a new generation vaccine in order to enhance the immunogenicity of the conventional yeast derived HBV vaccine.

Rabies VLPs adjuvanted with saponin-based liposomes induce enhanced immunogenicity mediated by neutralizing antibodies in cattle, dogs and cats.

We carried out an investigation on rabies virus-like particles (RV-VLPs) expressed in HEK293 cells using serum free medium. These RV-VLPs were formulated with two different adjuvants in order to analyse the enhancement of the triggered immune response and its stability. In experiments in mice, RV-VLPs showed an enhanced humoral immune response when injected with adjuvant, in contrast to the obtained for the RV-VLPs without adjuvant addition. Besides, higher titers of neutralizing antibodies were induced when RV-VLPs were formulated with LipoSap® in comparison with the obtained with Alhydrogel®. At the same time, the positive effect of this adjuvant in vaccine's potency and stability was demonstrated, showing that LipoSap® significantly increases the value obtained in NIH efficiency test for rabies vaccine, and proving that this value is maintained after 15 months storage at 4 °C. Further, we showed that RV-VLPs induces an immune response based on neutralizing antibodies when cat, dogs and bovines were vaccinated with only one dose of RV-VLPs. These results demonstrated that this vaccine candidate could be applied for the prevention of rabies in pets as well as for the control of paralytic rabies in cattle.

Optimization and validation of a blocking ELISA for quantitation of anti-rabies immunoglobulins in multispecies sera.

We developed a fast, rabies virus-free, in vitro method, based on a blocking ELISA (bELISA), to detect and accurately quantify anti-rabies glycoprotein antibodies in serum of several animal species. In this method, purified rabies virus-like particles (VLPs) are used as antigen to coat the plates, while the presence of specific rabies immunoglobulins is revealed through blocking the recognition of these VLPs by a biotinylated monoclonal antibody. A quality by design approach was carried out in order to optimize the method performance, improving the sensitivity and, thereby, reducing the limit of detection of this assay. After the method validation, we confirmed that the bELISA method is able to detect a concentration of 0.06 IU/mL rabies immunoglobulins, titer lower than the 0.5 IU/mL cutoff value established as indication for correct vaccination. Further, we assessed the correlation between bELISA, the MNT, and the Platelia methods, confirming the accuracy of this new assay. On the other hand, precision was evaluated, obtaining acceptable repeatability and intermediate precision values, showing that this bELISA could be proposed as a potential alternative method, replacing the gold standard techniques in vaccination schemes and becoming a routine control technique within regional rabies surveillance programs.

A simplified roller bottle platform for the production of a new generation VLPs rabies vaccine for veterinary applications.

Rabies is a neglected disease with an estimated annual mortality of 55,000 human deaths, affecting mainly low-income countries. Over 95% of these cases result from virus transmission through the bite of infected dogs and for this reason there is a real need for a cheap and effective rabies veterinary vaccine to be used in mass vaccination campaigns. In this work, we describe the establishment of a simple platform for the production of a virus-like particles based rabies vaccine using mammalian cells and roller bottles as culture system. Adherent cells were cultured during more than 15 days and VLPs were continuously produced and secreted to the culture supernatant. Immunogenicity and protective efficacy of VLPs were tested through rabies virus neutralizing antibody test and NIH potency test. These viral particles induced high titer of long lasting neutralizing antibodies and protected mice against active virus challenge. Therefore, this development represents a promising platform for the production of a new generation and virus-free rabies vaccine candidate for veterinary applications.

Knowledge of the Avalanche Victim Resuscitation Checklist and Utility of a Standardized Lecture in Italy.

INTRODUCTION: To explore baseline knowledge about avalanche guidelines and the Avalanche Victim Resuscitation Checklist (AVReCh) in Italy and the knowledge acquisition from a standardized lecture. METHODS: Standardized lecture material discussing AVReCh was presented during 8 mountain medicine courses from November 2014 to April 2016 in different regions of Italy. To determine the knowledge acquisition from the lecture, a pre- and postlecture survey was utilized. RESULTS: A total of 193 surveys were analyzed. More than 50% of the participants had never participated in lectures/courses on avalanche guidelines, and less than 50% of the participants knew about the AVReCh before the lecture. The correct temporal sequence of reportable information in the basic life support section of the AVReCh was selected by 40% of the participants before the lecture and by 75% after the lecture (P<0.001). Within subgroups analysis, most groups saw significant improvement in performance (P<0.05). The selection of the correct burial time increased from 36 to 84% (P<0.05). CONCLUSIONS: Health care providers and mountain rescue personnel are not widely aware of avalanche guidelines. The standardized lecture significantly improved knowledge of the principles of avalanche management related to core AVReCh elements. However, the effect that this knowledge acquisition has on avalanche victim survival or adherence to the AVReCh in the field is yet to be determined.

Development of Rabies Virus-Like Particles for Vaccine Applications: Production, Characterization, and Protection Studies.

Rabies is a viral infection of the central nervous system for which vaccination is the only treatment possible. Besides preexposure, vaccination is highly recommended for people living in endemic areas, veterinarians, and laboratory workers. Our group has developed rabies virus-like particles (RV-VLPs) with immunogenic features expressed in mammalian cells for vaccine applications. In this chapter the methods to obtain and characterize a stable HEK293 cell line expressing RV-VLPs are detailed. Further, analytical ultracentrifugation steps to purify the obtained VLPs are developed, as well as western blot, dynamic light scattering, and immunogold electron microscopy to analyze the size, distribution, shape, and antigenic conformation of the purified particles. Finally, immunization protocols are described to study the immunogenicity of RV-VLPs.