Our results are in agreement with these findings in that the highest levels of neutralizing antibody titers were elicited by DNA-VLP heterologous prime-boost immunization regardless of which influenza FliC and VLP types were used

Our results are in agreement with these findings in that the highest levels of neutralizing antibody titers were elicited by DNA-VLP heterologous prime-boost immunization regardless of which influenza FliC and VLP types were used. Glycan masking around the immunodominant epitopes of surface immunogens can elicit more broadly neutralizing antibodies if immunodominant sites overlap with the sequence variable regions of antigenic sites. 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 obtaining may have value in terms of novel immunogen design for developing cross-protective H5N1 vaccines. Introduction Highly pathogenic avian influenza (HPAI) H5N1 viruses and their transmission capability from birds to humans have raised global concerns about a potential human pandemic, with new H5N1strains emerging and evolving. The World Health Organization (WHO) has classified recently isolated H5N1 viruses into 10 clades or sublineages, based on phylogenetic analysis of viral hemagglutinin (HA) sequences [1]. With the ongoing threat of an influenza pandemic arising from avian reservoirs, the development of broadly protective vaccines is particularly important. To date, such vaccines have been achieved such as using novel adjuvant formulations [2]. However, the inherent nature of antigenic changes in influenza viruses has not been sufficiently taken into account in immunogen designs for broadly protective H5N1 vaccines. One approach is usually to refocus antibody responses by designing immunogens that can preserve overall immunogen structure, but selectively mutate undesired antigenic sites that are highly variable (i.e., mutants that evade protective Rabbit Polyclonal to MARK2 immune responses), immunosuppressive (i.e., downregulate immune responses to infections), or cross-reactive (i.e., immune responses induce reactions to proteins resembling immunogen) [3]C[9]. By refocusing antibody responses, the Limonin immunogen design has been applied to HIV-1 vaccines- that is, hyperglycosylated HIV-1 gp120 immunogens have been used, with undesired epitopes masked by the selective incorporation of N-linked glycans [4], [6], [10]C[12]. This glycan-masking strategy has also been used in the design of vaccines aimed at enhancing Limonin antibody responses to a broad range of H3N2 intertypic viruses [13]. However, to date there are no reports for glycan-masking immunogens for H5N1 vaccines. DNA vaccines offer advantages in terms of genetic antigen design, manufacturing time, stability in the absence of Limonin cold chains and immunogenicity elicited by T cells via endogenerous antigen processing pathways [14]. The problem of low DNA immunogenicity in large animals and humans has been overcome through the use of novel delivery systems such as gene-guns and electroporation [14]. Furthermore, DNA vaccine-elicited immune responses can be augmented by heterologous prime-boost immunization regimens, in which booster doses use a different vaccine format made up of identical or comparable antigens. DNA vaccine prime-boost immunization strategies have been described for inactivated influenza viruses [15], [16], live-attenuated influenza viruses [17], recombinant adenoviruses [18], virus-like particles (VLPs) [19], [20] and recombinant subunit proteins in adjuvants [21]C[25]. Humans receiving H5 DNA vaccine priming followed by a booster Limonin with an inactivated H5N1 vaccine were found to enhance the protective antibody responses, and in some cases induce hemagglutinin stem-specific neutralizing antibodies [16]. For this study we designed a hyperglycosylated HA vaccine using N-linked glycan masking on highly variable sequences in the HA1 globular head. Priming with hyperglycosylated HA DNA vaccine followed by a booster of flagellin-containing influenza virus-like particles (FliC-VLPs) in mice. FliC is usually a Toll-like receptor 5 (TLR-5) ligand and has been widely used for vaccine design, for its ability to induce the innate immune effectors, like cytokine and nitric oxide, e.g. induction of macrophage nitric oxide production [26] and activation of interleukin-1 receptor-associated kinase [27], thereby stimulating the activation of adaptive immune response. We previously reported that this influenza VLP can be fabricated by M2 fusion with FliC to improve and broaden the elicited neutralizing antibodies against homologous and heterologous HPAI H5N1 viruses [28]. We hope these findings have value in terms of novel immunogen design for developing cross-protective H5N1 vaccines. Materials and Methods DNA-HA vaccine vector construction Complimentary DNA (cDNA) from the HA gene of the A/Thailand/1(KAN-1)/2004/H5N1 influenza virus (clade 1) was generously provided by Prasert Auewarakul of Siriraj Hospital, Thailand. A full-length HA sequence was inserted into a pcDNA?3.1(+) vector (Invitrogen) via a KpnI/NotI cut site. The HA-containing plasmid was transfected into 293 cells using Turbofect reagent (Fermentas). At 48 h post-transfection, cell lysates were collected by centrifugation at 5000 rpm for 10 minutes, and HA expression was analyzed by Western blotting using anti-H5N1 HA antibodies (ab21297; Abcam). HA glycosylation patterns and trypsin treatment To characterize HA glycosylation patterns, 293 cells were harvested following transfection with DNA-HA vectors for 48 h. Cell lysates were treated with Endo H or PNGase F for 2 h at 37C, and HA glycosylation patterns were determined by Western blotting. For trypsin treatment, cell lysates were incubated with trypsin for 30 min on ice, and HA0 cleavage into HA1 and HA2 was confirmed by Western blotting. FliC-VLP preparation FliC-VLPs.