Characterization of Influenza H5N1 Nucleocapsid Protein for Potential Vaccine Design

Avian influenza, subtype H5N1, causes occasional but serious infections in humans and efforts to produce vaccines against this strain continue. Current influenza vaccines are prophylactic and utilize the two major antigens, hemagglutinin and neuraminidase. Nucleocapsid protein (NP) is an attractive alternative antigen because it is highly conserved across all influenza strains, has been shown to increase the rate of viral clearance, and potential therapeutic vaccines would elicit cytotoxic T lymphocyte responses in an infected person. The NP antigen from H5N1 was characterized using a variety of physico-chemical methods to gain insights into both the biological and physical properties of the antigen which are important from a regulatory viewpoint when considering therapeutic vaccines. Results obtained to date show that NP is relatively unstable and indicate that the conformation of the H5N1 NP antigen is highly dependent upon purification procedure, buffer conditions, pH and the presence or absence of RNA. These factors will need to be clearly defined and taken into consideration when manufacturing and regulating NP vaccine preparations.

Evaluation of a rehabilitation support service after acute stroke: Feasibility and patient/carer benefit

Background: Stroke survivors returning home after discharge from hospital and their carers require support to meet their rehabilitation needs (independence in Activities of Daily Living, exercise, psychosocial support). Voluntary or charitable care providers may be able to address some of these needs. Objective: To explore the feasibility of delivering and evaluating enhanced support to stroke survivors and their carers, with a Rehabilitation Support Worker (RSW). Methods: 16 consecutive stroke survivors and their carers were included. All participants received usual hospital care. Seven of these patients and their carers were also allocated an RSW from a charitable care provider. The RSW accompanied therapy training sessions with the patient, carer and therapist in hospital. On discharge, the RSW visited the patient and carer at home over the initial 6 week post-discharge period to support them in practising rehabilitation skills. Patient function (Barthel Index) and patient/carer confidence were independently assessed at discharge (Week 0). The above assessments and patient/carer mood (GHQ-12) and Carer Giver Strain were also assessed at Weeks 1, 6 and 12. RSWs were interviewed for their views about the service. Results: Participants’ functional ability at Week 1 post-discharge was significantly higher in the RSW group. At 6 and 12 weeks post-discharge, functional ability was not significantly different between groups. Carers in the intervention group were less confident at all time points, however, this was not significant. There was no significant effect on carer strain or well-being. Interviews with RSWs highlighted areas of their training that could be enhanced and the need for greater clarity as to their role. Conclusions: The results showed that a definitive trial of rehabilitation support is feasible. A number of obstacles however would need to be overcome including: difficulty in identifying suitable patients, clarity of the RSW role, and appropriate training content.

Monitoring Neutralization Property Change of Evolving Hantaan and Seoul Viruses with a Novel Pseudovirus-Based Assay

The Hantaan virus(HTNV)and Seoul virus(SEOV)mutants have accumulated over time.It is important to determine whether their neutralizing epitopes have evolved,thereby making the current vaccine powerless.However,it is impossible to determine by using traditional plaque reduction neutralization test(PRNT),because it requires large numbers of live mutant strains.Pseudovirus-based neutralization assays(PBNA)were developed by employing vesicular stomatitis virus(VSV)backbone incorporated with HTNV or SEOV glycoproteins(VSVDG*-HTNVG or VSVDG*-SEOVG).56 and 51 single amino acid substitutions of glycoprotein(GP)in HTNV and SEOV were selected and introduced into the reference plasmid.Then the mutant pseudoviruses were generated and tested by PBNA.The PBNA results were highly correlated with PRNT ones with R2 being 0.91 for VSVDG*-HTNVG and 0.82 for VSVDG*-SEOVG.53 HTNV mutant pseudoviruses and 46 SEOV mutants were successfully generated.Importantly,by using PBNA,we found that HTNV or SEOV immunized antisera could neutralize all the corresponding 53 HTNV mutants or the 46 SEOV mutants respectively.The novel PBNA enables us to closely monitor the effectiveness of vaccines against large numbers of evolving HTNV and SEOV.And the current vaccine remains to be effective for the naturally occurring mutants.

The molecular determinants governing the immunogenicity of Japanese encephalitis live attenuated vaccines

In the course of isolating the attenuated Japanese encephalitis vaccine SA14-14-2,two attenuated strains SA14-9-7 and SA14-5-3 were also obtained that elicited low antibody responses in humans(o10%and 62%,respectively)and exerted much weaker immune protection in animal challenge experiments.However,the reason for these differences remains unknown.In order to understand why SA14-14-2 is superior to SA14-9-7 and SA14-5-3,we employed a reverse genetics method to identify the key mutations in the virus genome that determine the immunogenicity of live attenuated Japanese encephalitis viruses.We first sequenced the full genomic sequences of SA14-9-7 and SA14-5-3 and found mutations that changed four amino-acid base pairs when compared to the envelope gene of SA14-14-2.We mutated the genome of SA14-14-2 to generate these mutations both singly(E-177,E-264,E-279 and E-315)and in combination(E-177/264,E-279/315 and E-177/264/279/315)and tested these mutants along with parental strains SA14-14-2,SA14-9-7 and SA14-5-3 for their immunogenicity in vivo.When mice were immunized with SA14-9-7 and SA14-5-3,lower levels of neutralizing antibodies were generated compared with the immune response to SA14-14-2.Furthermore,SA14-5-3 was more immunogenic than SA14-9-7,which replicated the results previously seen in humans.Point mutations E-177,E-264,E-279 and E-315 diminished the immunogenicity of SA14-14-2 with E-264 and E-315,contributing the most to this phenotype.The mutant rJEV(E-177/E-264/E-279/E-315)containing all four point mutations exhibited the lowest immunogenicity with a seroconversion rate of 0 at an inoculation dose of 103 PFU(plaque-forming unit).We have identified the key amino acids in the envelope protein that account for the superior immunogenicity of SA14-14-2.