Nanoemulsion W805EC improves immune responses upon intranasal delivery of an inactivated pandemic H1N1 influenza vaccine

Abstract

Currently available influenza vaccines provide suboptimal protection. In order to improve the quality of protective immune responses elicited following vaccination, we developed an oil-in-water nanoemulsion (NE)-based adjuvant for an intranasally-delivered inactivated influenza vaccine. Using a prime-boost vaccination regimen, we show that intranasal vaccines containing the W(80)5EC NE elicited higher titers of serum hemagglutination inhibiting (HAI) antibody and influenza-specific IgG and IgA titers compared to vaccines that did not contain the NE. Similarly, vaccines containing the W(80)5EC NE resulted in higher influenza-specific IgA levels in the bronchoalveolar lavage (BAL) fluid and nasal wash when compared to vaccines formulated without NE. The higher antibody titers in mice immunized with the NE-containing vaccines correlated with reduced viral loads in the lungs and nasal turbinates following a high dose viral challenge. Mice immunized with vaccines containing the W(80)5EC NE also showed a reduction in body weight loss following challenge compared to mice immunized with equivalent vaccines produced without NE. Taken together, our results show that the W(80)5EC NE substantially improves the magnitude of protective influenza-specific antibody responses and is a promising mucosal adjuvant for influenza vaccines and vaccines against other mucosal pathogens.

Figure 1. Screening NE formulations for mucosal adjuvant activity

A. Mice were immunized and boosted with intranasal vaccines prepared in the indicated nanoemulsion formulations or PBS only as a control. The titer of influenza-specific serum IgG in vaccinated mice four weeks after the booster immunization was determined by indirect ELISA. B. Mice were immunized and boosted with intranasal vaccines prepared in the indicated nanoemulsion formulations or PBS as a control. The relative amounts of influenza-specific IgA in bronchoalveolar lavage fluid collected from vaccinated mice four weeks after the booster immunization was determined by indirect ELISA. Bronchoalveolar lavage fluids were tested using a 1:2 dilution. Error bars represent the standard deviation; 3-5 mice per group. Statistically significant differences (P ≤ 0.05) are indicated.

Figure 2. Intranasal vaccines prepared in the W805EC NE increase hemagglutination inhibiting antibody levels

Mice were intranasally administered vaccines containing the indicated dose of β-propiolactone-inactivated H1N1 influenza virus in the W805EC NE or phosphate buffered saline (PBS) according to the time line in Supplementary Figure 1. Sera, collected 56 days following the first immunization, were serially diluted and tested for the ability to inhibit influenza virus-mediated agglutination of turkey red blood cells. Data points represent antibody titers in individual mice. Horizontal bars indicate the geometric mean. HAI: hemagglutination inhibiting.

Figure 3. Intranasal vaccines prepared in the W805EC NE increase influenza-specific serum IgG and IgA levels

Mice were administered vaccines containing the indicated dose of β-propiolactone-inactivated pandemic (H1N1) 2009 influenza virus according to the time line in Supplementary Figure 1. Intranasally delivered vaccines were prepared in the W805EC NE or phosphate buffered saline (PBS), and intramuscularly delivered vaccines were prepared in Alum or PBS. Sera were collected 56 days after the first immunization and influenza-specific IgG (left panel) and IgA (right panel) titers were determined by indirect ELISA. Data points represent antibody titers in individual mice. Horizontal bars indicate the geometric mean. Statistically significant differences (P ≤ 0.05) are indicated.

Figure 4. Intranasal vaccines prepared in the W805EC NE increase mucosal influenzaspecific IgG and IgA levels

Mice were administered vaccines containing the indicated dose of β-propiolactone-inactivated H1N1 influenza virus according to the time line in Supplementary Figure 1. Intranasally delivered vaccines were prepared in the W805EC NE or phosphate buffered saline (PBS), and intramuscularly delivered vaccines were prepared in Alum or PBS. Bronchoalveolar lavage fluid (A) and nasal wash (B) were collected 56 days after the first immunization and influenza-specific IgG (left panels) and IgA (right panels) titers were determined by indirect ELISA. Data points represent antibody titers in individual mice. Horizontal bars indicate the geometric mean. Statistically significant differences (P ≤ 0.05) are indicated.

Figure 5. Intranasal vaccines prepared in the W805EC NE decrease viral loads in respiratory organs following high-dose viral challenge

Mice were intranasally administered vaccines containing the indicated dose of β-propiolactone-inactivated H1N1 influenza virus in the W805EC NE or phosphate buffered saline (PBS) according to the time line in Supplementary Figure 1. Four weeks after the second administration of vaccine, mice were challenged with 100LD50 of mouse-adapted A/Wisc/09 virus. Lungs (A) and nasal turbinates (B) were collected at day 3 (left panels) and day 6 (right panels) following challenge and virus titer was determined using a plaque assay. Data points represent viral loads per gram of tissue (PFU/g) in individual mice. Horizontal bars indicate the mean. Statistically significant differences (P ≤ 0.05) are indicated.

Figure 6. Intranasal vaccines prepared in the W805EC NE reduce morbidity following viral challenge

Mice were intranasally or intramuscularly administered with vaccines containing 5×10⁶ PFU (A) or 5×10⁵ PFU (B) β-propiolactone-inactivated pandemic (H1N1) 2009 A/Wisc/09 virus according to the time line in Supplementary Figure 1. Vaccines were prepared in the W805EC NE or phosphate buffered saline (PBS) for intranasal delivery, or in Alum for intramuscular delivery. Cohorts of mice received the W805EC NE with no viral antigen (0 PFU groups) as a control. Four weeks after the second administration of vaccine, mice were challenged with 100LD50 of mouse adapted A/Wisc/09 virus and survival and weight loss was monitored daily for two weeks. Error bars represent standard deviation. A repeated measures ANOVA was used to calculate P values for overall weight loss curves of mice receiving vaccine that included NE and those receiving vaccine without NE. Statistically significant differences (P ≤ 0.05) are indicated.