Vaxfectin adjuvant improves antibody responses of juvenile rhesus macaques to a DNA vaccine encoding the measles virus hemagglutinin and fusion proteins

Abstract

DNA vaccines formulated with the cationic lipid-based adjuvant Vaxfectin induce protective immunity in macaques after intradermal (i.d.) or intramuscular (i.m.) delivery of 0.5 to 1 mg of codon-optimized DNA encoding the hemagglutinin (H) and fusion (F) proteins of measles virus (MeV). To characterize the effect of Vaxfectin at lower doses of H+F DNA, rhesus macaques were vaccinated twice with 20 μg of DNA plus Vaxfectin i.d., 100 μg of DNA plus Vaxfectin i.d., 100 μg of DNA plus Vaxfectin i.m. or 100 μg of DNA plus phosphate-buffered saline (PBS) i.m. using a needleless Biojector device. The levels of neutralizing (P = 0.036) and binding (P = 0.0001) antibodies were higher after 20 or 100 μg of DNA plus Vaxfectin than after 100 μg of DNA plus PBS. Gamma interferon (IFN-γ)-producing T cells were induced more rapidly than antibody, but were not improved with Vaxfectin. At 18 months after vaccination, monkeys were challenged with wild-type MeV. None developed rash or viremia, but all showed evidence of infection. Antibody levels increased, and IFN-γ- and interleukin-17-producing T cells, including cells specific for the nucleoprotein absent from the vaccine, were induced. At 3 months after challenge, MeV RNA was detected in the leukocytes of two monkeys. The levels of antibody peaked 2 to 4 weeks after challenge and then declined in vaccinated animals reflecting low numbers of bone marrow-resident plasma cells. Therefore, Vaxfectin was dose sparing and substantially improved the antibody response to the H+F DNA vaccine. This immune response led to protection from disease (rash/viremia) but not from infection. Antibody responses after challenge were more transient in vaccinated animals than in an unvaccinated animal.

Fig 1

Antibody responses to vaccination. Monkeys were vaccinated on day 0 and boosted 4 weeks later with codon-optimized DNA plasmids expressing the MeV H and F proteins. The vaccine was delivered either intradermally (i.d.) or intramuscularly (i.m.) either naked (PBS) or formulated with Vaxfectin (Vax). (A) Reciprocal titers of neutralizing antibody as measured by 50% plaque reduction of Chicago-1infection of Vero cells. The data are plotted as geometric means ± the standard errors of the mean (SEM). A dashed line indicates the generally accepted protective levels. Comparison of 100 μg of DNA i.m. with or without Vaxfectin (P = 0.0356, Student t test). (B) Enzyme immunoassay of plasma (1:100) IgG binding to MeV lysate-coated wells. The data are expressed as optical density + the SEM (P = 0.0001, one-way ANOVA). (C) Plasma taken 48 weeks after vaccination or 135 days after infection with Bilthoven compared for neutralization of Chicago-1 infection of Vero cells (interaction with CD46; black bars) and neutralization of Bilthoven infection of Vero/hSLAM cells (interaction with CD150; gray bars).

Fig 2

IFN-γ T cell responses to vaccination. PBMCs from monkeys vaccinated as described above were assessed for production of IFN-γ in response to stimulation with pooled overlapping peptides from the H protein (A and B) and the F protein (C and D). The results are presented as SFCs/10⁶ PBMCs for individual monkeys (A and C) and as averaged values ± the SEM for each vaccine group (B and D).

Fig 3

IL-4 T cell responses to vaccination. At 2 weeks after the first dose of vaccine, PBMCs were assessed for IL-4 production after stimulation with overlapping peptides from the H protein and the F protein. The results are presented as SFCs/10⁶ PBMCs.

Fig 4

Protection from viremia after challenge. PBMCs collected at days 3, 7, 10, and 14 after intratracheal challenge with 10⁴ TCID₅₀ of the Bilthoven strain of WT MeV were cocultivated with Vero/hSLAM cells and read for cytopathic effect. None of the vaccinated animals developed viremia or rash after challenge, while the unvaccinated animal developed both rash and viremia. The data are presented as TCID₅₀/10⁶ PBMCs.

Fig 5

B cell responses after challenge. Plasma, PBMCs, and bone marrow (BM) cells were collected from vaccinated and unvaccinated monkeys after challenge with WT MeV. (A) MeV-specific IgG in plasma as determined by enzyme immunoassay. The data are presented as the mean of OD values + the SEM. (B) Numbers of plasmablasts producing MeV-specific antibody present in circulation 7 and 10 days after challenge. ASCs, antibody-secreting cells. (C) Numbers of plasma cells in bone marrow producing MeV-specific antibody before (d0, 17 months after vaccine boost) and after (105 and 140 days) challenge.

Fig 6

IFN-γ T cell responses after challenge. Vaccinated and unvaccinated monkeys were challenged intratracheally with the Bilthoven WT strain of MeV. PBMCs were examined ex vivo without stimulation for production of IFN-γ (A and B) and after stimulation with pools of overlapping peptides from the H (C and D), F (E and F), and N (G and H) proteins. The data are presented as SFCs per 10⁶ PBMCs for individual animals (A, C, E, and G) and as averages plus the SEM for each vaccine group (B, D, F, and H). For stimulated cells (C to H), the numbers of SFCs present without stimulation (A and B) have been subtracted.

Fig 7

IL-17 T cell responses after challenge. After intratracheal challenge, PBMCs were examined ex vivo without stimulation (A and B) and after stimulation with MeV-infected cell lysate (C and D) for IL-17 production. The data are presented as SFCs/10⁶ PBMCs for individual animals (A and C) and as means + the SEM for each group (B and D). For stimulated cells (C), the numbers of SFCs present without stimulation (A) have been subtracted.