Virus-like particles as powerful vaccination strategy against human viruses.

Nowadays, viruses are not only seen as causative agents of viral infectious diseases but also as valuable research materials for various biomedical purposes, including recombinant protein production. When expressed in living or cell-free expression systems, viral structural proteins self-assemble into virus-like particles (VLPs). Mimicking the native form and size of viruses and lacking the genetic material, VLPs are safe and highly immunogenic and thus can be exploited to develop antiviral vaccines. Some vaccines based on VLPs against various infectious pathogens have already been licenced for human use and are available in the commercial market, the latest of which is a VLP-based vaccine to protect against the novel Coronavirus. Despite the success and popularity of VLP subunit vaccines, many more VLPs are still in different stages of design, production, and approval. There are still many challenges that require to be addressed in the future before this surface display system can be widely used as an effective vaccine strategy in combating infectious diseases. In this review, we highlight the use of structural viral proteins to produce VLPs, emphasising their intrinsic properties, structural classification, and main expression host systems. We also compiled the recent scientific literature about VLP-based vaccines to underline the recent advances in their application as a vaccine strategy for preventing and fighting virulent human pathogens. Finally, we presented the key challenges and possible solutions for VLP-based vaccine production.

Viral Protein VP1 Virus-like Particles (VLP) of CVB4 Induces Protective Immunity against Lethal Challenges with Diabetogenic E2 and Wild Type JBV Strains in Mice Model.

Several epidemiological studies demonstrated that coxsackievirus B4 (CVB4) causes viral pancreatitis and can ultimately result in type 1 diabetes mellitus (T1D). Prevention of CVB4 infection is therefore highly desirable. There is currently no vaccine or antiviral therapeutic reagent in clinical use. VLP are structurally similar to native virus particles and therefore are far better immunogens than any other subunit vaccines. Many studies have shown the potential of capsid protein VP1 on providing protective effects from different viral strains. In this study, we contributed towards the development of a CVB4 VLP-based vaccine from the total protein VP1 of the diabetogenic CVB4E2 strain and assessed whether it could induce a protective immunity against both the wild-type CVB4JBV and the diabetogenic CVB4E2 strains in mice model. Serum samples, taken from mice immunized with VLP, were assayed in vitro for their anti-CVB4 neutralizing activity and in vivo for protective activity. We show that VLP vaccine generates robust immune responses that protect mice from lethal challenges. Results demonstrate that CVB4 VP1 capsid proteins expressed in insect cells have the intrinsic capacity to assemble into non-infectious VLP, which afforded protection from CVB4 infection to mice when used as a vaccine.

The introduction of mutations in the wild type coxsackievirus B3 (CVB3) IRES RNA leads to different levels of in vitro reduced replicative and translation efficiencies.

Coxsackievirus B3 (CVB3) is a principal causative agent of viral myocarditis, meningitis and pancreatitis. There is no vaccine available for clinical use. It has been demonstrated that the primary molecular determinant of virulence phenotype is located in the 5' UTR of the viral genome. Translation initiation of CVB3 RNA is directed by the IRES element situated in the 5'UTR. In the present study, we analyse the effects of single point mutations introduced in different positions in the domain V of the IRES RNA of CVB3 wild type. We characterize in vitro virus replicative capacitiy and translation efficiency and we test in vivo virulence of different CVB3 mutants produced by the introduction of different mutations in the domain V of IRES by site-directed mutagenesis to abolish its structure. Our results demonstrate that all RNA mutants display different levels of decreased replication and translation initiation efficiency in vitro. The translation defect was correlated with significant reduced viral titer of mutant particles in comparison with the wild type. When inoculated in mice, mutant viruses were checked for inflammation and necrosis.In vitro and in vivo Findings strongly suggest that the most attenuated mutant strain could be considered a candidate for live-attenuated CVB3 vaccine.

A Single Mutation in the Cryptic AUG (cAUG) Affects In Vitro Translation and Replication Efficiencies and In Vivo Virulence of Coxsackievirus B3 (CVB3).

The 5'UTR of the genomic RNA of CVB3, unusually long and rich on highly structured secondary structure, contains a conserved cis acting RNA element named the cryptic AUG (cAUG), where the cellular 48S complex is formed. In this study, we investigate the role of this cAUG in CVB3 translation, replication, and virulence. Mutant viral sub-genomic replicon RNA was constructed by site-directed mutagenesis. We characterize in vitro translation and replication efficiencies and in vivo virulence of a cAUG mutant in comparison with wild-type strain. UV-cross-linking assay and Real-Time PCR were used, respectively, to detect binding host proteins and to quantify viral production. Secondary structures of domain containing the cAUG site were studied and compared. The results suggest that introduced mutation in the CVB3 5'UTR affects in vitro and ex vivo viral translation which cannot be rescued by compensatory mutations. A reduced interaction of the La and PCBP2 translation initiation factors with cAUG residue of mutant was revealed. Decreasing production of viral mutant RNA was also demonstrated. Furthermore, secondary structure prediction reveals changes in the ribosome binding sites of the cAUG moiety of mutant sense strand RNA and no alterations in the structure of wild type, suggesting that cAUG mutation specifically affects the secondary structure of the sense RNA strand. Taken together, AUG integrity influences the efficiency of ribosome recruitment through IRES element and the capacity of replication.

Lethal Mutagenesis of RNA Viruses and Approved Drugs with Antiviral Mutagenic Activity.

In RNA viruses, a small increase in their mutation rates can be sufficient to exceed their threshold of viability. Lethal mutagenesis is a therapeutic strategy based on the use of mutagens, driving viral populations to extinction. Extinction catastrophe can be experimentally induced by promutagenic nucleosides in cell culture models. The loss of HIV infectivity has been observed after passage in 5-hydroxydeoxycytidine or 5,6-dihydro-5-aza-2'-deoxycytidine while producing a two-fold increase in the viral mutation frequency. Among approved nucleoside analogs, experiments with polioviruses and other RNA viruses suggested that ribavirin can be mutagenic, although its mechanism of action is not clear. Favipiravir and molnupiravir exert an antiviral effect through lethal mutagenesis. Both drugs are broad-spectrum antiviral agents active against RNA viruses. Favipiravir incorporates into viral RNA, affecting the G→A and C→U transition rates. Molnupiravir (a prodrug of ß-d-N4-hydroxycytidine) has been recently approved for the treatment of SARS-CoV-2 infection. Its triphosphate derivative can be incorporated into viral RNA and extended by the coronavirus RNA polymerase. Incorrect base pairing and inefficient extension by the polymerase promote mutagenesis by increasing the G→A and C→U transition frequencies. Despite having remarkable antiviral action and resilience to drug resistance, carcinogenic risks and genotoxicity are important concerns limiting their extended use in antiviral therapy.

Phylogenetic analysis of the neuraminidase segment gene of Influenza A/H1N1 strains isolated from Monastir Region (Tunisia) during the 2017-2018 outbreak.

Influenza A/H1N1 is widely considered to be a very evolutionary virus causing major public health problems. Since the pandemic of 2009, there has been a rapid rise in human Influenza virus characterization. However, little data is available in Tunisia regarding its genetic evolution. In light of this fact, our paper aim is to genetically characterize the Neuraminidase, known as the target of antiviral inhibitors, in Tunisian isolates circulating in Monastir region during 2017-2018. In total of 31 positive Influenza A/H1N1 detected by multiplex real-time PCR, RT-PCR of neuraminidase was performed. Among the 31 positive samples, 7 samples representing fatal and most severe cases were conducted for sequencing and genetic analysis. The results thus obtained showed genetic evolution of the A/H1N1 neuraminidase between 2009 and 2010 and 2018-2019 outbreaks. All Tunisian isolates were genetically related to the recommended vaccine strain with a specific evolution. Moreover, the phylogenetic analysis demonstrated that France and especially Italian strains were the major related strains. Interestingly, our results revealed a specific cluster of Tunisian isolates where two intragroup were evolved in correlation with the severity and the fatalities cases. From the outcome of our investigation, this study confirms the genetic evolution of the Influenza A virus circulating in Tunisia and gives a preliminary analysis for a better comprehension of new emerging Tunisian strain's virulence and thus, a more appropriate monitoring of Influenza virus A/H1N1 during each round of outbreaks. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11756-021-00723-y.

Characterization of Coxsackievirus B4 virus-like particles VLP produced by the recombinant baculovirus-insect cell system expressing the major capsid protein.

Coxsackievirus B4 (CV-B4) is suspected to be an environmental factor that has the intrinsic capacity to damage the pancreatic beta cells and therefore causes insulitis and type 1 diabetes (T1D). Although vaccination against CV-B4 could reduce the incidence of this chronic auto-immune disease, there is currently no therapeutic reagent or vaccine in clinical use. By the employment of the Bac-to-Bac® vector system to express the major viral capsid protein, we contributed towards the development of a CV-B4 vaccine by producing CV-B4 virus-like particles (VLPs) from recombinant baculovirus in infected insect cells. In fact Western blot and Immunofluorescence analysis detected the viral protein 1 (VP1) in the cells resulting from the construction of a recombinant bacmid DNA carrying the key immunogenic protein then transfected in the insect cells. Sucrose gradient ultracentrifugation fractions of the infected cell lysates contained the recombinant protein and the electron microscopy demonstrated the presence of VLPs in these sucrose fractions. This study clearly shows for the first time the expression of CVB4 VP1 structure protein alone can form VLPs in the baculovirus-infected insect cell keeping conserved both characteristics and morphology.

In vitro-reduced translation efficiency of coxsackievirus B3 Sabin3-like strain is correlated to impaired binding of cellular initiation factors to viral IRES RNA.

Coxsackievirus B3 (CVB3) causes viral myocarditis and can ultimately result in dilated cardiomyopathy. There is no vaccine available for clinical use. Translation initiation of CVB3 RNA is directed by an internal ribosome entry site within the 5'-untranslated region. We have previously described that Sabin3-like mutation (U(473) to C) introduced in CVB3 genome led to a defective mutant with a serious reduction in translation efficiency. In the present study, we analyzed, in vitro, the effect of the Sabin3-like mutation on the binding affinity of RNA domain V to some standard translation initiation factors: eIF4G, eIF3b, and eIF4B by filter-binding assays and UV-crosslink assays. We have demonstrated that this single-nucleotide exchange impairs the binding affinity of these cellular factors within the mutant RNA. These data indicate how this decisive Sabin3-like mutation mediates viral translation attenuation. Taken together, these findings strongly suggest that the mutant strain could be considered a candidate for an attenuated CVB3 vaccine.

Multiplex RT-PCR and indirect immunofluorescence assays for detection and subtyping of human influenza virus in Tunisia.

Influenza viruses are negative stranded segmented RNA viruses belonging to Orthomyxoviridae family. They are classified into three types A, B, and C. Type A influenza viruses are classified into subtypes according to the antigenic characters of the surface glycoproteins: hemagglutinin (H) and neuraminidase (N). The aim of the present study is to develop a fast and reliable multiplex RT-PCR technique for detecting simultaneously the subtypes A/H1N1 and A/H3N2 of influenza virus. Our study included 398 patients (mean age 30.33 ± 19.92 years) with flu or flu-like syndromes, consulting physicians affiliated with collaborating teams. A multiplex RT-PCR detecting A/H1N1 and A/H3N2 influenza viruses and an examination by indirect immunofluorescence (IFI) were performed. In the optimized conditions, we diagnosed by IFI a viral infection in 90 patients (22.6 %): 85 cases of influenza type A, four cases of influenza type B, and only one case of coinfection with types A and B. An evaluation of the technique was performed on 19 clinical specimens positive in IFI, and we detected eight cases of A/H3N2, five cases of A/H1N1, one case of influenza virus type A which is not an H1N1 nor H3N2, and five negative cases. Multiplex RT-PCR is a sensitive technique allowing an effective and fast diagnosis of respiratory infections caused by influenza viruses in which the optimization often collides with problems of sensibility.

Nucleotide sequences of IRES domains IV and V of natural ECHO virus type 11 isolates with different replicative capacity phenotypes.

ECHO viruses (ECV) belong to the enterovirus genus of the Picornaviridae family and are the most frequently isolated from clinical and environmental samples. They are responsible for a wide variety of clinical syndromes involving most organs of the human body. We previously postulated that some of the variations in the recognition of ECHO virus type 11 (ECV 11) strains by a group specific monoclonal antibody (Mab) which we have studied could be explained by variations in their replicative capacity in cell culture and variations within the 5' nontranslated region (5' NTR) of their genomes. To support this hypothesis, the replicative capacity in cell culture and the nucleotide sequences of domains IV and V of the IRES of the genome of five ECV11 strains (the Gregory reference strain and four wild isolates) were determined, and analysed. Our results indicate that the replicative capacity of wild ECV11 isolates studied by one-step growth cycle in both HEp-2 and Vero cell cultures showed variations among strains in comparison with the Gregory reference strain. The clinical ECV11 strains replicated as well as the reference strain, however environmental strains displayed a phenotype with a significant reduction of replication. The sequences of ECV 11 strains showed significant conservation with that of the poliovirus (PV1) Mahoney strain The comparative examination of the predicted secondary structures revealed, that the nucleotide variations did not affect the secondary structure of stem-loop structure IV and V in the IRES element, however differences were especially observed in the apical stem region (nucleotides 483 to 509) of the domain V of the ECV11 strains and resulted in modification of the central stem structure.

Epidemiological study of non-polio enterovirus neurological infections in children in the region of Monastir, Tunisia.

The epidemiological, virological, and clinical syndromes of 86 children younger than 13 years suspected of having neurological diseases and admitted to Fattouma Bourguiba Hospital of Monastir from January 2002 to November 2003 were analyzed. The presence of enterovirus was studied in cerebrospinal fluid (CSF) samples by reverse transcription-polymerase chain reaction (RT-PCR) and by isolation on cell culture. Sixty-one (71%) specimens were positive by RT-PCR, whereas 45 (52.3%) were detected by cell culture. Eighty percent (n = 36) of cultured enteroviruses were identified, whereas 20% were untyped. Echoviruses were isolated most frequently, with 32 cases (71.1%) in children. Echovirus 6 was the most commonly identified serotype (22.2%), followed by echovirus 13 (20%). The highest incidence on neurological infection (19.6%) occurred in children less than 6 months of age. The infected children were predominantly male (62.3%). Enteroviruses were detected in all the period of the year with the highest rate in the spring and summer months. Aseptic meningitis was the most commonly diagnosed disease (49%).