Development of Novel Single-Chain Antibodies against the Hydrophobic HPV-16 E5 Protein

The human papilloma virus type 16 infects genital mucosa with high prevalence in the oncogenesis of cervical and oropharyngeal cancers. The E5 protein of this virus is a small hydrophobic protein, whose expression generally decreases as the infection progresses to malignancy. These characteristics point to a role of E5 in the establishment of HPV infection and the initiation into cell transformation. The study of the HPV-16 E5 functions has been hindered because of the lack of antibodies. Detection is very difficult because of its hydrophobic nature, membrane location, and very low levels of expression. Thus, the objective of this study was to select single-chain antibodies against the full size E5 protein, which was coexpressed with maltose-binding protein. We report that the E5 protein was recognized by the antibody and was validated in W12 cells by fluorescent microscopy, including a colocalization with one of its host substrates. The use of this antibody could increase our knowledge about the functions of the oncogenic HPV-16 E5 protein during the earliest stages of keratinocyte infection in human.

Genomic non-coding regions reveal hidden patterns of mumps virus circulation in Spain, 2005 to 2015

Background Since mumps vaccination was introduced in 1981 in Spain, the incidence of the disease has dropped significantly. However, cyclic epidemic waves and outbreaks still occur, despite high vaccination coverage. The World Health Organization (WHO) recommends genotyping to trace the pattern of mumps virus (MuV) circulation. Genotype H was predominant in Spain, but was replaced in 2005 by genotype G which has subsequently remained dominant. Of the small hydrophobic protein gene sequences, 78% are identical and belong to the MuVi/ Sheffield.GBR.1.05/[G]-variant. Aim: Our study aimed to investigate whether the circulation of MuV strains in Spain was continuous after the emergence of genotype G in 2005. Method: We obtained 46 samples from Spanish patients infected with MuVi/Sheffield.GBR.1.05/[G] during two epidemic waves and analysed them using new molecular markers based on genomic non-coding regions (NCRs) that discriminate subvariants of this virus strain. Results: Phylogenetic analyses of the nucleoprotein–phosphoprotein and matrix protein–fusion protein NCR indicated strain replacement after a drop in incidence in 2009, which had not been detectable by SH sequencing. Clustering of sequences from patients epidemiologically linked in the same outbreak suggests a potential use for these NCRs in outbreak characterisation. Conclusion: We suggest to consider their use in conjunction with the SH gene in the future WHO recommendations for MuV epidemiological surveillance.

A Recombinant Respiratory Syncytial Virus Vaccine Candidate Attenuated by a Low-Fusion F Protein Is Immunogenic and Protective against Challenge in Cotton Rats

ABSTRACTAlthough respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants, a safe and effective vaccine is not yet available. Live-attenuated vaccines (LAVs) are the most advanced vaccine candidates in RSV-naive infants. However, designing an LAV with appropriate attenuation yet sufficient immunogenicity has proven challenging. In this study, we implemented reverse genetics to address these obstacles with a multifaceted LAV design that combined the codon deoptimization of genes for nonstructural proteins NS1 and NS2 (dNS), deletion of the small hydrophobic protein (ΔSH) gene, and replacement of the wild-type fusion (F) protein gene with a low-fusion RSV subgroup B F consensus sequence of the Buenos Aires clade (BAF). This vaccine candidate, RSV-A2-dNS-ΔSH-BAF (DB1), was attenuated in two models of primary human airway epithelial cells and in the upper and lower airways of cotton rats. DB1 was also highly immunogenic in cotton rats and elicited broadly neutralizing antibodies against a diverse panel of recombinant RSV strains. When vaccinated cotton rats were challenged with wild-type RSV A, DB1 reduced viral titers in the upper and lower airways by 3.8 log10total PFU and 2.7 log10PFU/g of tissue, respectively, compared to those in unvaccinated animals (P< 0.0001). DB1 was thus attenuated, highly immunogenic, and protective against RSV challenge in cotton rats. DB1 is the first RSV LAV to incorporate a low-fusion F protein as a strategy to attenuate viral replication and preserve immunogenicity.IMPORTANCERSV is a leading cause of infant hospitalizations and deaths. The development of an effective vaccine for this high-risk population is therefore a public health priority. Although live-attenuated vaccines have been safely administered to RSV-naive infants, strategies to balance vaccine attenuation with immunogenicity have been elusive. In this study, we introduced a novel strategy to attenuate a recombinant RSV vaccine by incorporating a low-fusion, subgroup B F protein in the genetic background of codon-deoptimized nonstructural protein genes and a deleted small hydrophobic protein gene. The resultant vaccine candidate, DB1, was attenuated, highly immunogenic, and protective against RSV challenge in cotton rats.