Investigation of Avian Influenza H5N6 Virus-like Particles as a Broad-Spectrum Vaccine Candidate against H5Nx Viruses.

Highly pathogenic avian influenza (HPAI) clade 2.3.4.4 viruses have been reported to be the source of infections in several outbreaks in the past decades. In a previous study, we screened out a broad-spectrum virus strain, H5N6-Sichuan subtype, by using a lentiviral pseudovirus system. In this project, we aimed to investigate the potential of H5N6 virus-like particles (VLPs) serving as a broad-spectrum vaccine candidate against H5Nx viruses. We cloned the full-length M1 gene and H5, N6 genes derived from the H5N6-Sichuan into pFASTBac vector and generated the VLPs using the baculovirus-insect cell system. H5N6 VLPs were purified by sucrose gradient centrifugation, and the presence of H5, N6 and M1 proteins was verified by Western blot and SDS-PAGE. The hemagglutination titer of H5N6 VLPs after purification reached 5120 and the particle structure remained as viewed by electron microscopy. The H5N6 VLPs and 293T mammalian cell-expressed H5+N6 proteins were sent for mice immunization. Antisera against the H5+N6 protein showed 80 to 320 neutralizing antibody titers to various H5Nx pseudoviruses. In contrast, H5N6 VLPs not only elicited higher neutralizing antibody titers, ranging from 640 to 1280, but also induced higher IL-2, IL-4, IL-5, IFN-γ and TNF production, thus indicating that H5N6 VLPs may be a potential vaccine candidate for broad-spectrum H5Nx avian influenza vaccines.

Comparison of Immunogenicity and Safety between a Single Dose and One Booster Trivalent Inactivated Influenza Vaccination in Patients with Chronic Kidney Disease: A 20-Week, Open-Label Trial.

BACKGROUND: Non-dialysis-dependent chronic kidney disease (CKD-ND) patients are recommended to receive a one-dose influenza vaccination annually. However, studies investigating vaccine efficacy in the CKD-ND population are still lacking. In this study, we aimed to evaluate vaccine efficacy between the one-dose and two-dose regimen and among patients with different stages of CKD throughout a 20-week follow-up period. METHODS: We conducted a single-center, non-randomized, open-label, controlled trial among patients with all stages of CKD-ND. Subjects were classified as unvaccinated, one-dose, and two-dose groups (4 weeks apart) after enrollment. Serial changes in immunological parameters (0, 4, 8, and 20 weeks after enrollment), including seroprotection, geometric mean titer (GMT), GMT fold-increase, seroconversion, and seroresponse, were applied to evaluate vaccine efficacy. RESULTS: There were 43, 84, and 71 patients in the unvaccinated, one-dose, and two-dose vaccination groups, respectively. At 4-8 weeks after vaccination, seroprotection rates in the one- and two-dose group for H1N1, H3N2, and B ranged from 82.6-95.8%, 97.4-100%, and 73.9-100%, respectively. The concomitant seroconversion and GMT fold-increases nearly met the suggested criteria for vaccine efficacy for the elderly population. Although the seroprotection rates for all of the groups were adequate, the seroconversion and GMT fold-increase at 20 weeks after vaccination did not meet the criteria for vaccine efficacy. The two-dose regimen had a higher probability of achieving seroprotection for B strains (Odds ratio: 3.5, 95% confidence interval (1.30-9.40)). No significant differences in vaccine efficacy were found between early (stage 1-3) and late (stage 4-5) stage CKD. CONCLUSIONS: The standard one-dose vaccination can elicit sufficient protective antibodies. The two-dose regimen induced a better immune response when the baseline serum antibody titer was low. Monitoring change in antibody titers for a longer duration is warranted to further determine the current vaccine strategy in CKD-ND population.

Assessing the application of a pseudovirus system for emerging SARS-CoV-2 and re-emerging avian influenza virus H5 subtypes in vaccine development.

BACKGROUND: Highly pathogenic emerging and re-emerging viruses continuously threaten lives worldwide. In order to provide prophylactic prevention from the emerging and re-emerging viruses, vaccine is suggested as the most efficient way to prevent individuals from the threat of viral infection. Nonetheless, the highly pathogenic viruses need to be handled in a high level of biosafety containment, which hinders vaccine development. To shorten the timeframe of vaccine development, the pseudovirus system has been widely applied to examine vaccine efficacy or immunogenicity in the emerging and re-emerging viruses. METHODS: We developed pseudovirus systems for emerging SARS coronavirus 2 (SARS-CoV-2) and re-emerging avian influenza virus H5 subtypes which can be handled in the biosafety level 2 facility. Through the generated pseudovirus of SARS-CoV-2 and avian influenza virus H5 subtypes, we successfully established a neutralization assay to quantify the neutralizing activity of antisera against the viruses. RESULTS: The result of re-emerging avian influenza virus H5Nx pseudoviruses provided valuable information for antigenic evolution and immunogenicity analysis in vaccine candidate selection. Together, our study assessed the potency of pseudovirus systems in vaccine efficacy, antigenic analysis, and immunogenicity in the vaccine development of emerging and re-emerging viruses. CONCLUSION: Instead of handling live highly pathogenic viruses in a high biosafety level facility, using pseudovirus systems would speed up the process of vaccine development to provide community protection against emerging and re-emerging viral diseases with high pathogenicity.

Mapping Enterovirus A71 Antigenic Determinants from Viral Evolution.

UNLABELLED: Human enterovirus A71 (EV-A71) belongs to the Enterovirus A species in the Picornaviridae family. Several vaccines against EV-A71, a disease causing severe neurological complications or even death, are currently under development and being tested in clinical trials, and preventative vaccination programs are expected to start soon. To characterize the potential for antigenic change of EV-A71, we compared the sequences of two antigenically diverse genotype B4 and B5 strains of EV-A71 and identified substitutions at residues 98, 145, and 164 in the VP1 capsid protein as antigenic determinants. To examine the effects of these three substitutions on antigenicity, we constructed a series of recombinant viruses containing different mutation combinations at these three residues with a reverse genetics system and then investigated the molecular basis of antigenic changes with antigenic cartography. We found that a novel EV-A71 mutant, containing lysine, glutamine, and glutamic acid at the respective residues 98, 145, and 164 in the VP1 capsid protein, exhibited neutralization reduction against patients' antisera and substantially increased virus binding ability to human cells. These observations indicated that this low-neutralization-reactive EV-A71 VP1-98K/145Q/164E mutant potentially increases viral binding ability and that surveillance studies should look out for these mutants, which could compromise vaccine efficacy. IMPORTANCE: Emerging and reemerging EV-A71 viruses can cause severe neurological etiology, primarily affecting children, especially around Asia-Pacific countries. We identified a set of mutations in EV-A71 that both reduced neutralization activity against humoral immunity in antisera of patients and healthy adults and greatly increased the viral binding ability to cells. These findings provide important insights for EV-A71 antigenic determinants and emphasize the importance of continuous surveillance, especially after EV-A71 vaccination programs begin.