Broader Neutralizing Antibodies against H5N1 Viruses Using Prime-Boost Immunization of Hyperglycosylated Hemagglutinin DNA and Virus-Like Particles

Abstract

Background Highly pathogenic avian influenza (HPAI) H5N1 viruses and their transmission capability from birds to humans have raised global concerns about a potential human pandemic. The inherent nature of antigenic changes in influenza viruses has not been sufficiently taken into account in immunogen designs for broadly protective HPAI H5N1 vaccines. Methods We designed a hyperglycosylated HA vaccine using N-linked glycan masking on highly variable sequences in the HA1 globular head. Immunization of these hyperglycosylated HA DNA vaccines followed by a flagellin-containing virus-like particle booster in mice was conducted to evaluate neutralizing antibody responses against various clades of HPAI H5N1 viruses. Results We introduced nine N-X-S/T motifs in five HA1 regions: 83NNT, 86NNT, 94NFT, 127NSS, 138NRT, 156NTT, 161NRS, 182NDT, and 252NAT according to sequence alignment analyses from 163 HPAI H5N1 human isolates. Although no significant differences of anti-HA total IgG titers were found with these hyperglycosyalted HA compared to the wild-type control, the 83NNT and 127NSS mutants elicited significantly potent cross-clade neutralizing antibodies against HPAI H5N1 viruses. Conclusions This finding may have value in terms of novel immunogen design for developing cross-protective H5N1 vaccines.

DNA Vaccine that Targets Hemagglutinin to MHC Class II Molecules Rapidly Induces Antibody-Mediated Protection against Influenza

“…The discrepancy might be explained by the current study employing only a single immunization, a low injection volume, and a low DNA dose. By contrast, previous studies by others describe protection first after several immunizations (6,7,10), larger volumes (7,9,10,56,57), and larger amounts of DNA (9,(56)(57)(58). Another factor that may explain the discrepancy is that we have used a truncated HA in which most of the transmembrane and intracellular parts have been deleted.…”

“…However, the induction of adequate immune responses against influenza require large doses of DNA even in mice, and often in combination with adjuvants or several booster injections (4)(5)(6)(7)(8)(9)(10). Also, DNA vaccines often have a tendency of skewing immune responses toward T cell immunity, whereas Abs are of major importance in influenza prevention.…”

Unmasking Stem-Specific Neutralizing Epitopes by Abolishing N-Linked Glycosylation Sites of Influenza Virus Hemagglutinin Proteins for Vaccine Design

“…Female 6-to 8-week-old BALB/c mice (5 per group) purchased from the Taiwan National Laboratory Animal Center were immunized intramuscularly (i.m.) twice with 20 g of either WT or stem glycan mutant proteins of H5N1-rHA or pH1N1-rHA plus 10 g CpG and 10% PELC emulsion over a 3-week interval (19,20). Serum samples were collected at week 5, and the sera were inactivated at 56°C for 30 min to destroy complement prior to performing neutralization assays (both H5N1 pseudotyped particle [H5pp] assay and plaque reduction neutralization test [PRNT]); splenocytes were harvested and isolated at week 7.…”

“…To neutralize antibodies against the H5N1 virus, neutralization titers were quantified as reduced luciferase expression levels using the lentivirus H5N1 pseudotyped particle (H5pp) assay as previously described (19,20). Fifty microliters of H5pp at 100 50% tissue culture infective doses (TCID 50 ) was incubated with 50 l of serially diluted antisera (1:10 starting dilution) for 1 h at 37°C, followed by the addition of 1.5 ϫ 10 4 MDCK cells per well.…”

“…Another approach uses stem antigens that lack the globular head as soluble trimeric proteins (17), or perhaps incorporating them into ferritin nanoparticles for immunization purposes to elicit stem-specific antibodies (18). The third approach uses the glycan shielding on the variable regions in the HA globular head to redirect the immune responses to the more conserved HA stem region (19)(20)(21). Several reports also indicate that the HA stem-based influenza vaccines provide cross-subtype protection against diverse group 1 strains but not against diverse group 2 strains (15)(16)(17)(18).…”