Improved protection against avian influenza H5N1 virus by a single vaccination with virus-like particles in skin using microneedles

Abstract

To develop a more effective vaccination method against H5N1 virus, we investigated the immunogenicity and protective efficacy after skin vaccination using microneedles coated with influenza virus-like particles containing hemagglutinin derived from A/Vietnam/1203/04 H5N1 virus (H5 VLPs). A single microneedle vaccination of mice with H5 VLPs induced increased levels of antibodies and provided complete protection against lethal challenge without apparent disease symptoms. In contrast, intramuscular injection with the same vaccine dose showed low levels of antibodies and provided only partial protection accompanied by severe body weight loss. Post-challenge analysis suggested that improved protection was associated with lower lung viral titers and enhanced generation of recall antibody secreting cells by microneedle vaccination. Thus, this study provides evidence that skin delivery of H5 VLP vaccines using microneedles designed for self-administration induces improved protection compared to conventional intramuscular immunization.

Fig. 1. Immunogenicity of influenza H5 VLP vaccination in the skin using microneedles

(a) A microneedle coated with H5 VLPs before and after dissolution in PBS. A microneedle is shown as observed by bright field microscopy after coating with influenza H5 VLPs for skin vaccination (i) and after dissolution of the coating from the microneedle in PBS (ii). Bar=250 μm. (b) H5 HA-specific IgG titers at weeks 3 and 7 after a single vaccination. Titers of antibodies specific to inactivated H5 A/Vietnam/1203/04 virus are expressed as the highest dilution of sera having a value of optical density at 450 nm (OD450) greater than the mean plus 2 standard deviations of similarly diluted naïve sera as described previously (Quan et al., 2007). (c) H5 HA-specific isotype antibodies as presented in OD450 values of 100× diluted sera at week 7. BALB/c mice (n=11 per group) were immunized once with H5 VLPs using microneedles (MN), intramuscular injection (IM), or uncoated placebo microneedles (Mock). Bars indicate means ± S.E.M. (d) Hemagglutination inhibition (HI) titers determined at week 7 after vaccination. Asterisk indicates significance between MN and IM groups (**; p<0.01, *; p<0.05, Student's 2-tailed t-test).

Fig. 2. Protection by a single-dose microneedle vaccination with H5 VLPs

Groups of BALB/c mice that had been vaccinated (Mock, IM, MN) were intranasally inoculated with a lethal dose (20×LD₅₀) of wild type A/Vietnam/1203/04 (H5N1) virus at week 20 post vaccination. (a) Daily mean body weight (n=5 out of 11 vaccinated mice). Significant morbidity indicated by greater than 15% weight loss as well as ruffling fur and inactivity between days 6-10 post challenge was observed in the IM group. In contrast, the MN group had no weight loss. (b) Daily proportion of mice alive (n=5 out of 11 vaccinated mice).

Fig. 3. Lung viral titers and virus-specific antibody-secreting cell responses

Six out of eleven vaccinated mice (Mock, IM, MN) were sacrificed for analysis of lung viral titers and antibody secreting cell responses at day 4 post challenge as described (Quan et al., 2007). (a) Lung viral titers were determined by a plaque assay with lung homogenate obtained from 6 individual mice. (b) Recall antibody secreting cell responses of spleen cells. Antibody levels were presented as concentrations in culture supernatants (expressed in ng/ml). Values are means ± S.E.M. of individual mice (n=6) after in vitro culture. Asterisk indicates significance between MN and IM groups (*; p<0.05, **; p<0.01, Student's 2-tailed t-test).