Rotavirus vaccine-derived cases in Belgium: Evidence for reversion of attenuating mutations and alternative causes of gastroenteritis.

Since the introduction of live-attenuated rotavirus vaccines in Belgium in 2006, surveillance has routinely detected rotavirus vaccine-derived strains. However, their genomic landscape and potential role in gastroenteritis have not been thoroughly investigated. We compared VP7 and VP4 nucleotide sequences obtained from rotavirus surveillance with the Rotarix vaccine sequence. As a result, we identified 80 vaccine-derived strains in 5125 rotavirus-positive infants with gastroenteritis from 2007 to 2018. Using both viral metagenomics and reverse transcription qPCR, we evaluated the vaccine strains and screened for co-infecting enteropathogens. Among the 45 patients with known vaccination status, 39 were vaccinated and 87% received the vaccine less than a month before the gastroenteritis episode. Reconstruction of 30 near complete vaccine-derived genomes revealed 0-11 mutations per genome, with 88% of them being non-synonymous. This, in combination with several shared amino acid changes among strains, pointed at selection of minor variant(s) present in the vaccine. We also found that some of these substitutions were true revertants (e.g., F167L on VP4, and I45T on NSP4). Finally, co-infections with known (e.g., Clostridioides difficile and norovirus) and divergent or emerging (e.g., human parechovirus A1, salivirus A2) pathogens were detected, and we estimated that 35% of the infants likely had gastroenteritis due to a 'non-rotavirus' cause. Conversely, we could not rule out the vaccine-derived gastroenteritis in over half of the cases. Continued studies inspecting reversion to pathogenicity should monitor the long-time safety of live-attenuated rotavirus vaccines. All in all, the complementary approach with NGS and qPCR provided a better understanding of rotavirus vaccine strain evolution in the Belgian population and epidemiology of co-infecting enteropathogens in suspected rotavirus vaccine-derived gastroenteritis cases.

Crystallization of a nonreplicating rotavirus vaccine candidate.

Nonreplicating rotavirus vaccine (NRRV) candidates are being developed with the aim of serving the needs of developing countries. A significant proportion of the cost of manufacturing such vaccines is the purification in multiple chromatography steps. Crystallization has the potential to reduce purification costs and provide new product storage modality, improved operational flexibility, and reduced facility footprints. This communication describes a systematic approach for the design of the crystallization of an NRRV candidate, VP8 subunit proteins fused to the P2 epitope of tetanus toxin, using first-principles models and preliminary experimental data. The first-principles models are applied to literature data to obtain feasible crystallization conditions and lower bounds for nucleation and growth rates. Crystallization is then performed in a hanging-drop vapor diffusion system, resulting in the nucleation and growth of NRRV crystals. The crystals obtained in a scaled-up evaporative crystallization contain proteins truncated in the P2 region, but have no significant differences with the original samples in terms of antibody binding and overall conformational stability. These results demonstrate the promise of evaporative crystallization of the NRRV.

Holistic process development to mitigate proteolysis of a subunit rotavirus vaccine candidate produced in Pichia pastoris by means of an acid pH pulse during fed-batch fermentation.

To meet the challenges of global health, vaccine design and development must be reconsidered to achieve cost of goods as low as 15¢ per dose. A new recombinant protein-based rotavirus vaccine candidate derived from non-replicative viral subunits fused to a P2 tetanus toxoid CD4(+) T cell epitope is currently under clinical development. We have sought to simplify the existing manufacturing process to meet these aims. To this end, we have taken a holistic process development approach to reduce process complexity and costs while producing a product with the required characteristics. We have changed expression system from Escherichia coli to Pichia pastoris, to produce a secreted product, thereby reducing the number of purification steps. However, the presence of proteases poses challenges to product quality. To understand the effect of fermentation parameters on product quality small-scale fermentations were carried out. Media pH and fermentation duration had the greatest impact on the proportion of full-length product. A novel acidic pH pulse strategy was used to minimize proteolysis, and this combined with an early harvest time significantly increased the proportion of full-length material (60-75%). An improved downstream process using a combination of CIEX and AIEX to further reduce proteases, resulted in maintaining product quality (95% yield).

Clearance of porcine circovirus and porcine parvovirus from porcine-derived pepsin by low pH inactivation and cation exchange chromatography.

The contamination of oral rotavirus vaccines by porcine circovirus (PCV) raised questions about potential PCV contamination of other biological products when porcine trypsin or pepsin is used in production process. Several methods can be potentially implemented as a safety barrier when animal derived trypsin or pepsin is used. Removal of PCV is difficult by the commonly used viral filters with the pore size cutoff of approximately 20 nm because of the smaller size of PCV particles that are around 17 nm. It was speculated that operating the chromatography step at a pH higher than pepsin's low pI, but lower than pIs, of most viruses would allow the pepsin to flow through the resin and be recovered from the flow through pool whilst the viruses would be retained on the resin. In this study, we investigated low pH inactivation of viruses including PCV Type 1 (PCV1) and PCV1 removal by cation exchange chromatography (CEX) in the presence of pepsin. Both parvovirus and PCV1 could be effectively inactivated by low pH and PCV1 could be removed by POROS 50HS CEX. The POROS 50HS method presented in this article is helpful for designing other CEX methods for the same purpose and not much difference would be expected for similar product intermediates and same process parameters. While the effectiveness needs to be confirmed for specific applications, the results demonstrate that both low pH (pH 1.7) and CEX methods were successful in eliminating PCV1 and thus either can be considered as an effective virus barrier.

Characterizing and Minimizing Aggregation and Particle Formation of Three Recombinant Fusion-Protein Bulk Antigens for Use in a Candidate Trivalent Rotavirus Vaccine.

In a companion paper, the structural integrity, conformational stability, and degradation mechanisms of 3 recombinant fusion-protein antigens comprising a non-replicating rotavirus (NRRV) vaccine candidate (currently being evaluated in early-stage clinical trials) are described. In this work, we focus on the aggregation propensity of the 3 NRRV antigens coupled to formulation development studies to identify common frozen bulk candidate formulations. The P2-VP8-P[8] antigen was most susceptible to shaking and freeze-thaw-induced aggregation and particle formation. Each NRRV antigen formed aggregates with structurally altered protein (with exposed apolar regions and intermolecular ß-sheet) and dimers containing a non-native disulfide bond. From excipient screening studies with P2-VP8-P[8], sugars or polyols (e.g., sucrose, trehalose, mannitol, sorbitol) and various detergents (e.g., Pluronic F-68, polysorbate 20 and 80, PEG-3350) were identified as stabilizers against aggregation. By combining promising additives, candidate bulk formulations were optimized to not only minimize agitation-induced aggregation, but also particle formation due to freeze-thaw stress of P2-VP8-P[8] antigen. Owing to limited material availability, stabilization of the P2-VP8-P[4] and P2-VP8-P[6] was confirmed with the lead candidate P2-VP8-P[8] formulations. The optimization of these bulk NRRV candidate formulations is discussed in the context of subsequent drug product formulations in the presence of aluminum adjuvants.

Recombinant Subunit Rotavirus Trivalent Vaccine Candidate: Physicochemical Comparisons and Stability Evaluations of Three Protein Antigens.

Although live attenuated Rotavirus (RV) vaccines are available globally to provide protection against enteric RV disease, efficacy is substantially lower in low- to middle-income settings leading to interest in alternative vaccines. One promising candidate is a trivalent nonreplicating RV vaccine, comprising 3 truncated RV VP8 subunit proteins fused to the P2 CD4+ epitope from tetanus toxin (P2-VP8-P[4/6/8]). A wide variety of analytical techniques were used to compare the physicochemical properties of these 3 recombinant fusion proteins. Various environmental stresses were used to evaluate antigen stability and elucidate degradation pathways. P2-VP8-P[4] and P2-VP8-P[6] displayed similar physical stability profiles as function of pH and temperature while P2-VP8-P[8] was relatively more stable. Forced degradation studies revealed similar chemical stability profiles with Met1 most susceptible to oxidation, the single Cys residue (at position 173/172) forming intermolecular disulfide bonds (P2-VP8-P[6] was most susceptible), and Asn7 undergoing the highest levels of deamidation. These results are visualized in a structural model of the nonreplicating RV antigens. The establishment of key structural attributes of each antigen, along with corresponding stability-indicating methods, have been applied to vaccine formulation development efforts (see companion paper), and will be utilized in future analytical comparability assessments.

Effect of Aluminum Adjuvant and Preservatives on Structural Integrity and Physicochemical Stability Profiles of Three Recombinant Subunit Rotavirus Vaccine Antigens.

A nonreplicating rotavirus vaccine (NRRV) containing 3 recombinant fusion proteins adsorbed to aluminum adjuvant (Alhydrogel [AH]) is currently in clinical trials. The compatibility and stability of monovalent NRRV antigen with key components of a multidose vaccine formulation were examined using physicochemical and immunochemical methods. The extent and strength of antigen-adjuvant binding were diminished by increasing phosphate concentration, and acceptable levels were identified along with alternate buffering agents. Addition of the preservative thimerosal destabilized AH-adsorbed P2-VP8-P[8] as measured by differential scanning calorimetry. Over 3 months at 4°C, AH-adsorbed P2-VP8-P[8] was stable, whereas at 25°C and 37°C, instability was observed which was greatly accelerated by thimerosal addition. Loss of antibody binding (enzyme-linked immunosorbent assay) correlated with loss of structural integrity (differential scanning calorimetry, fluorescence spectroscopy) with concomitant nonnative disulfide bond formation (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and Asn deamidation (liquid chromatography -mass spectrometry peptide mapping). An alternative preservative (2-phenoxyethanol) showed similar antigen destabilization. Due to limited availability, only key assays were performed with monovalent P2-VP8-P[4] and P2-VP8-P[6] AH-adsorbed antigens, and varying levels of preservative incompatibility were observed. In summary, monovalent AH-adsorbed NRRV antigens stored at 4°C showed good stability without preservatives; however, future formulation development efforts are required to prepare a stable, preservative-containing, multidose NRRV formulation.

Stability of a monovalent rotavirus vaccine after exposure to different temperatures observed in KwaZulu-Natal, South Africa.

BACKGROUND: Rotavirus infection and its associated hospitalization of children less than 5 years old in middle- and low-income countries remains a public health challenge. We hypothesized that the Rotarix®potency is affected by non-optimal temperatures which translates into reduced vaccine effectiveness in these settings. OBJECTIVE: To assess the effect of non-optimal temperatures on the potency of the Rotarix® vaccine in South Africa. METHODS: Rotarix® vaccine was exposed to temperatures reflecting breaches in the cold chain. Vero cells (ATCC CCL-81) grown in a 24-well tissue culture plates were infected with Rotarix® vaccine viruses after exposure to non-optimal temperatures and the potency of the vaccine was determined using the plaque assay. RESULTS: Exposure of the Rotarix® vaccine to seasonal temperatures in KwaZulu-Natal for 6 hours and to extreme temperatures of 40oC for 72 hours as well as to -20°C and -80°C for 12 hours did not affect the potency of the vaccine beyond its expected standard of >7 x 105 PFU/ml. CONCLUSION: This study revealed that the Rotarix® vaccine remains potent even after exposure to non-optimal temperatures. However, this study only explored the effect of a constant 'adverse' temperature on vaccine potency and not the effect of temperature fluctuations.

Composition of gut microbiota and its influence on the immunogenicity of oral rotavirus vaccines.

The introduction of oral rotavirus vaccines (ORVVs) has led to a reduction in number of hospitalisations and deaths due to rotavirus (RV) infection. However, the efficacy of the vaccines has been varied with low-income countries showing significantly lower efficacy as compared to high-income countries. The reasons for the disparity are not fully understood but are thought to be multi-factorial. In this review article, we discuss the concept that the disparity in the efficacy of oral rotavirus vaccines between the higher and lower socio-economical countries could be due the nature of the bacteria that colonises and establishes in the gut early in life. We further discuss recent studies that has demonstrated significant correlations between the composition of the gut bacteria and the immunogenicity of oral vaccines, and their implications in the development of novel oral RV vaccines or redesigning the current ones for maximum impact.

Epidemiological features and genetic characterization of virus strains in rotavirus associated gastroenteritis in children of Odisha in Eastern India.

We have studied the clinical characteristics, severity and seasonality of rotavirus infection and prevalent genotypes in 652 non-rota vaccinated children in Odisha in eastern India. P genotypes were analysed for their association with host blood group antigens. P type of the virus is determined by the VP8* gene, and specific recognition of A - type of Histo - blood group antigen by P[14]VP8* has been reported. VP4, VP7 and VP6 genes of commonly identified G1P[8] strain were compared with genes of the same strain isolated from other parts of India, elsewhere and strains used for Rotarix and Rotateq vaccines. In 54.75% of children with gastroenteritis, rota virus was found. 9.65% of children had moderate, 78.07% severe, and 12.28% very severe disease as assessed using the Vesikari scoring system. The incidence of infection was highest during winter months. There was no association between any blood group and specific P genotypes. G1P[8] was the commonest cause of gastroenteritis, followed by G1P[11], G3P[8], G9P[8], G2P[4], G2P[6], G9P[4], G9P[11] and G1P[6]. Predominant G genotypes identified were G1 (72.9%), G9 (10.81%), G2 (8.10%) and G3 (8.10%). Sequence analysis of the VP7 gene, placed the G1P[8] strain in lineage 1 and of VP6 gene placed nine G1P[8] strains in subgroup II and one in subgroup I. The VP7 gene segment of two Odisha G1P[8] strains were found to cluster relatively close to the VP7 sequences of Rotarix vaccine. Antigenic differences were found with vaccine strains. Ten G1P[8] strains sequenced for the VP4 gene had 91-93% nucleotide and 92-96% amino acid identity with Rotateq vaccine P[8]). Rotarix vaccine VP4 had 89-91% nucleotide and 90-92% amino acid identity. Our findings indicate genetic variability of rotavirus strains circulating in the region and are significant, given the introduction of rota vaccination in the State.

Stability of heat stable, live attenuated Rotavirus vaccine (ROTASIIL®).

Vaccines currently available across the globe are stored and transported in a continuous cold-chain at 2-8°C or below -20°C. A temperature excursion outside this range affects the potency of the vaccines. Such vaccines need to be discarded leading wastage. The Rotavirus disease burden is predominantly reported in developing and low-income countries and therefore, has entered or poised to enter their national immunization programs. These countries already have several limitations for effective storage, maintenance and distribution of vaccines in a cold-chain and this introduction is expected to further stress this fragile ecosystem. To help mitigate the cold chain related issues, SIIPL has developed a thermostable rotavirus vaccine ROTASIIL® which can be stored at a temperature below 25°C for 36months, completely by-passing the standard 2-8°C cold storages. In addition it has the capability to withstand temperatures of 37°C and 40°C for 18months and short term exposure to 55°C. It can also tolerate a temperature shock of being thawed from an extreme cold temperature of -20°C to a high temperature of 42°C. The vaccine contains serotypes G1, G2, G3, G4 and G9 (UK-Bovine reassortant strains procured from National Institute of Health-USA). The vaccine is recently licensed in India.

Safety, Tolerability and Immunogenicity of Pentavalent Rotavirus Vaccine Manufactured by a Modified Process.

BACKGROUND: Rotavirus is the leading cause of severe diarrhea in infants and young children. The current formulation of pentavalent rotavirus vaccine (RV5) must be stored refrigerated at 2-8°C. A modified formulation of RV5 (RV5mp) has been developed with stability at 37°C for 7 days and an expiry extended to 36 months when stored at 2-8°C. METHODS: This study (ClinicalTrials.gov identifier: NCT01600092; EudraCT number: 2012-001611-23) evaluated the safety, tolerability and immunogenicity of RV5mp versus the currently marketed RV5 in infants. To maintain blinding, both vaccine formulations were stored refrigerated at 2-8°C for the duration of the study. Immunogenicity endpoints were (1) serum neutralizing antibody titers to human rotavirus serotypes G1, G2, G3, G4 and P1A[8] and (2) proportion of subjects with a ≥3-fold rise from baseline for serum neutralizing antibody to human rotavirus serotypes G1, G2, G3, G4 and P1A[8] and serum antirotavirus immunoglobulin A. RESULTS: The RV5mp group (n = 505) and RV5 group (n = 509) had comparable safety profiles. There were no deaths and no vaccine-related serious adverse events in this study. With respect to immunogenicity, RV5mp was noninferior compared with RV5. Serum neutralizing antibody responses by country and breast-feeding status were generally consistent with the overall results. CONCLUSIONS: RV5mp enhances storage requirements while maintaining the immunogenicity and safety profile of the currently licensed RV5. A vaccine that is stable at room temperature may be more convenient for vaccinators, particularly in places where the cold chain is unreliable, and ultimately will permit more widespread use.

Rotavirus vaccine stability in the aquatic environment.

AIMS: To evaluate the thermal and length of stability of the Rotaviruses (RV) vaccine (RotaTeq) in the aquatic environment. METHODS AND RESULTS: Surface freshwater, brackish and drinking water were spiked with RV vaccine strain and stored at 22 and 4°C. The virus infectivity and genome persistence were evaluated by plaque assay and RT-qPCR, respectively, up to 180 days. Infectious RV vaccine particles showed to be less stable in the brackish water matrix than in surface and drinking water either at 22 or 4°C. The estimated T90 values obtained by the linear regression model were 18, 55 and 59 days, respectively for brackish, surface and drinking water stored at 22°C and 68, 154 and 240 days at 4°C. As expected, the genome persistence showed to be less affected by length and temperature of storage in all the matrices evaluated. CONCLUSIONS: The evidence of high stability of the RV vaccine in water matrices reinforces the importance for surveillance of RV vaccines strains in the environment regarding the potential occurrence of unexpected infections and virus genomic reassortments. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of reassortants and the shedding of the live attenuated vaccine strains after vaccination can compromise the vaccine safety by introducing new viral variants in the environment.

Synthetic biology design to display an 18 kDa rotavirus large antigen on a modular virus-like particle.

Virus-like particles are an established class of commercial vaccine possessing excellent function and proven stability. Exciting developments made possible by modern tools of synthetic biology has stimulated emergence of modular VLPs, whereby parts of one pathogen are by design integrated into a less harmful VLP which has preferential physical and manufacturing character. This strategy allows the immunologically protective parts of a pathogen to be displayed on the most-suitable VLP. However, the field of modular VLP design is immature, and robust design principles are yet to emerge, particularly for larger antigenic structures. Here we use a combination of molecular dynamic simulation and experiment to reveal two key design principles for VLPs. First, the linkers connecting the integrated antigenic module with the VLP-forming protein must be well designed to ensure structural separation and independence. Second, the number of antigenic domains on the VLP surface must be sufficiently below the maximum such that a "steric barrier" to VLP formation cannot exist. This second principle leads to designs whereby co-expression of modular protein with unmodified VLP-forming protein can titrate down the amount of antigen on the surface of the VLP, to the point where assembly can proceed. In this work we elucidate these principles by displaying the 18.1 kDa VP8* domain from rotavirus on the murine polyomavirus VLP, and show functional presentation of the antigenic structure.

[Genotype of Rotavirus Vaccine Strain LLR in China is G10P[15]].

Rotavirus is the leading causal agent of severe acute gastroenteritis in children aged <5 years. A specific pharmacologic agent for the treatment of rotavirus-infected children is lacking. In China, only the Luo Tewei oral vaccine (Lanzhou Institute of Biological Products, Shanghai, China), which is produced from Lanzhou lamb rotavirus vaccine (LLR), is available. Studies have hypothesized that the genotype of LLR is G10P[12], To identify the genotype of LLR by reverse transcription-polymerase chain reaction, we showed that the VP7 and VP4 genotypes of LLR were G10 and P[15], respectively, based on sequencing, alignment and phylogenetic analyses. In conclusion, we identified the genotype of rotavirus strain LLR to be G10P[15].

Comparative characteristics of the VP7 and VP4 antigenic epitopes of the rotaviruses circulating in Russia (Nizhny Novgorod) and the Rotarix and RotaTeq vaccines.

Two live, attenuated rotavirus A (RVA) vaccines, Rotarix and RotaTeq, have been successfully introduced into national immunization programs worldwide. The parent strains of both vaccines were obtained more than 30 years ago. Nonetheless, only very limited data are available on the molecular similarity of the vaccine strains and their genetic relationships to the wild-type strains circulating within the territory of Russian Federation. In this study, we have determined the nucleotide sequences of the genes encoding the viral proteins VP7 and VP4 (the globular domain VP8*) of vaccine strains and natural isolates of rotaviruses in Nizhny Novgorod, Russia. The VP7 and VP4 proteins contain antigenic sites that are the main targets of neutralizing antibodies. Phylogenetic analysis based on VP4 and VP7 showed that the majority of the natural RVA isolates from Nizhny Novgorod and the vaccine strains belong to different clusters. Four amino acids within the VP7 antigenic sites were common in both the wild-type and vaccine strains. The largest number of amino acid differences was found between the vaccine strain Rotarix and the Nizhny Novgorod G2 strains (19 residues out of 29). From 3 to 5 amino acid differences per strain were identified in the antigenic sites of VP4 (domain VP8*) between wild-type strains and the vaccine RotaTeq, and 6-8 substitutions were found when they were compared with the vaccine strain Rotarix. For the first time, immunodominant T-cell epitopes of VP7 were analyzed, and differences in the sequences between the vaccine and the wild-type strains were found. The accumulation of amino acid substitutions in the VP7 and VP4 antigenic sites may potentially reduce the immune protection of vaccinated children from wild-type strains of rotavirus.

Detection of rotateq vaccine-derived, double-reassortant rotavirus in a 7-year-old child with acute gastroenteritis.

We describe a case of acute gastroenteritis in a schoolgirl associated with a detection of vaccine-derived, human-bovine double reassortant G1P[8] rotavirus, without any known contact with recently vaccinated infants. We propose that human-bovine double reassortant G1P[8] may be formed in RotaTeq-vaccinated infants and can occasionally cause gastroenteritis symptoms in vaccine recipients who may rarely transmit the virus to close contacts. The present case suggests that such viruses can remain stable in environment longer than 1 transmission cycle.

Investigation of a regulatory agency enquiry into potential porcine circovirus type 1 contamination of the human rotavirus vaccine, Rotarix: approach and outcome.

In January 2010, porcine circovirus type 1 (PCV1) DNA was unexpectedly detected in the oral live-attenuated human rotavirus vaccine, Rotarix (GlaxoSmithKline [GSK] Vaccines) by an academic research team investigating a novel, highly sensitive analysis not routinely used for adventitious agent screening. GSK rapidly initiated an investigation to confirm the source, nature and amount of PCV1 in the vaccine manufacturing process and to assess potential clinical implications of this finding. The investigation also considered the manufacturer's inactivated poliovirus (IPV)-containing vaccines, since poliovirus vaccine strains are propagated using the same cell line as the rotavirus vaccine strain. Results confirmed the presence of PCV1 DNA and low levels of PCV1 viral particles at all stages of the Rotarix manufacturing process. PCV type 2 DNA was not detected at any stage. When tested in human cell lines, productive PCV1 infection was not observed. There was no immunological or clinical evidence of PCV1 infection in infants who had received Rotarix in clinical trials. PCV1 DNA was not detected in the IPV-containing vaccine manufacturing process beyond the purification stage. Retrospective testing confirmed the presence of PCV1 DNA in Rotarix since the initial stages of its development and in vaccine lots used in clinical studies conducted pre- and post-licensure. The acceptable safety profile observed in clinical trials of Rotarix therefore reflects exposure to PCV1 DNA. The investigation into the presence of PCV1 in Rotarix could serve as a model for risk assessment in the event of new technologies identifying adventitious agents in the manufacturing of other vaccines and biological products.

Expression of human rotavirus chimeric fusion proteins from replicating but non disseminating adenovectors and elicitation of rotavirus-specific immune responses in mice.

The aim of this study was to evaluate the usefulness of replicating but non disseminating adenovirus vectors (AdVs) as vaccine vector using human rotavirus (HRV) as a model pathogen. HRV VP7, VP4, or VP4Δ (N-terminal 336 amino acids of VP4) structural proteins as well as the VP4Δ::VP7 chimeric fusion protein were expressed in mammalian cells when delivered with the AdVs. A preliminary experiment demonstrated that VP4Δ was able to induce a HRV-specific IgG response in BALB/c mice inoculated intramuscularly with AdVs expressing the rotaviral protein. Moreover, an AdV-prime/plasmid DNA-boost regimen of vectors resulted in VP4Δ-specific antibody (Ab) titers ~4 times higher than those obtained from mice immunized with AdVs alone. Subsequently, the various HRV protein-encoding AdVs were compared using the AdV-prime/plasmid DNA-boost regimen. Higher IgG and IgA responses to HRV were obtained when VP4Δ::VP7 fusion protein was used as an immunogen as compared to VP7 or VP4 alone or to a mix of both proteins delivered independently by AdVs. A synergetic effect in terms of Ab was obtained with VP4Δ::VP7. In conclusion, this study demonstrated for the first time the suitability of using replicating but non disseminating AdVs as vaccine vector and the VP4Δ::VP7 fusion protein as an immunogen for vaccination against HRV.

Molecular characterization analysis of the outer protein layer (VP7) from human rotavirus A genotype G1 isolate identified in Iran: implications for vaccine development.

The full open reading frame of the outer protein layer VP7 from an isolate of human rotavirus identified in 2010 in an Iranian child admitted to hospital with gastroenteritis was amplified from a clinical stool specimen and subjected to molecular characterization. Genetic and phylogenetic analyses indicated that the analyzed gene falls into the G1 genotype forming a sub-cluster with sequences recently identified in Iran and geographically distant countries. Such results were confirmed by protein sequence alignment, showing a highly conserved "G1-like?? amino acid sequence pattern within the known three main immunodominant regions. These results are extremely relevant in a perspective of vaccine development. Indeed, the present study confirms that the A group G1 genotype is the most prevalent Rotavirus circulating in Iran and supports the development of G1 genotype-based rotavirus vaccine for this country.