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. 2015 Oct 27;6(6):e01024-15.
doi: 10.1128/mBio.01024-15.

Inactivated Influenza Vaccine That Provides Rapid, Innate-Immune-System-Mediated Protection and Subsequent Long-Term Adaptive Immunity

Brendon Y Chua  1 , Chinn Yi Wong  2 , Edin J Mifsud  2 , Kathryn M Edenborough  2 , Toshiki Sekiya  2 , Amabel C L Tan  2 , Francesca Mercuri  2 , Steve Rockman  3 , Weisan Chen  4 , Stephen J Turner  2 , Peter C Doherty  2 , Anne Kelso  5 , Lorena E Brown  6 , David C Jackson  6
Affiliations

Inactivated Influenza Vaccine That Provides Rapid, Innate-Immune-System-Mediated Protection and Subsequent Long-Term Adaptive Immunity

Brendon Y Chua et al. mBio. .

Abstract

The continual threat to global health posed by influenza has led to increased efforts to improve the effectiveness of influenza vaccines for use in epidemics and pandemics. We show in this study that formulation of a low dose of inactivated detergent-split influenza vaccine with a Toll-like receptor 2 (TLR2) agonist-based lipopeptide adjuvant (R4Pam2Cys) provides (i) immediate, antigen-independent immunity mediated by the innate immune system and (ii) significant enhancement of antigen-dependent immunity which exhibits an increased breadth of effector function. Intranasal administration of mice with vaccine formulated with R4Pam2Cys but not vaccine alone provides protection against both homologous and serologically distinct (heterologous) viral strains within a day of administration. Vaccination in the presence of R4Pam2Cys subsequently also induces high levels of systemic IgM, IgG1, and IgG2b antibodies and pulmonary IgA antibodies that inhibit hemagglutination (HA) and neuraminidase (NA) activities of homologous but not heterologous virus. Improved primary virus nucleoprotein (NP)-specific CD8(+) T cell responses are also induced by the use of R4Pam2Cys and are associated with robust recall responses to provide heterologous protection. These protective effects are demonstrated in wild-type and antibody-deficient animals but not in those depleted of CD8(+) T cells. Using a contact-dependent virus transmission model, we also found that heterologous virus transmission from vaccinated mice to naive mice is significantly reduced. These results demonstrate the potential of adding a TLR2 agonist to an existing seasonal influenza vaccine to improve its utility by inducing immediate short-term nonspecific antiviral protection and also antigen-specific responses to provide homologous and heterologous immunity.

Importance: The innate and adaptive immune systems differ in mechanisms, specificities, and times at which they take effect. The innate immune system responds within hours of exposure to infectious agents, while adaptive immunity takes several days to become effective. Here we show, by using a simple lipopeptide-based TLR2 agonist, that an influenza detergent-split vaccine can be made to simultaneously stimulate and amplify both systems to provide immediate antiviral protection while giving the adaptive immune system time to implement long-term immunity. Both types of immunity induced by this approach protect against vaccine-matched as well as unrelated virus strains and potentially even against strains yet to be encountered. Conferring dual functionality to influenza vaccines is beneficial for improving community protection, particularly during periods between the onset of an outbreak and the time when a vaccine becomes available or in scenarios in which mass vaccination with a strain to which the population is immunologically naive is imperative.

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Figures

FIG 1
FIG 1
Rapid protective antiviral effects induced by inoculation with split virus vaccine formulated with R4Pam2Cys. (A) BALB/c mice (n = 4 to 5 per group) were inoculated via the intranasal route with PR8-derived split virus vaccine alone or combined with 5 nmol of R4Pam2Cys at 1 day (day −1), 3 days (day −3), or 7 days (day −7) prior to challenge with either 50 PFU of PR8 or 104.5 PFU of X31. (B) Lungs were harvested 5 days later and viral titers determined. Symbols represent the titers obtained from individual mice, and horizontal lines indicate the mean virus titers (± standard deviations [SD]) of the group. Groups are defined at the bottom of panel B. Asterisks (*) indicate P values of <0.05.
FIG 2
FIG 2
Inoculation with split virus vaccine formulated with R4Pam2Cys also provides rapid protection against lethal viral challenge in a TLR2-dependent manner. (A) BALB/c mice (n = 5 per group) were inoculated via the intranasal route with PR8-derived split virus vaccine alone or combined with 5 nmol of R4Pam2Cys 3 days prior to challenge with 500 PFU of PR8. (B) Lungs were harvested 5 days later, and viral titers were determined. Symbols represent the titers obtained from individual mice, and horizontal lines indicate the mean virus titers (± SD) for the group. C57BL/6 or TLR2−/− mice (n = 4 per group) were similarly inoculated 3 days prior to viral challenge and monitored daily for signs of illness and weight loss. Mice were killed when a humane endpoint was reached as characterized by >20% weight loss accompanied by signs of severe disease. (C and D) The mean body weight of mice is represented as a percentage of the original weight at the time of challenge as depicted in panel C, and survival over a 10-day period is shown in panel D. Asterisks (*) indicate P values of <0.05.
FIG 3
FIG 3
Induction of antibody and CD8+ T cell responses by vaccination. (A) Adaptive immunity induced by vaccination was studied by measuring antigen-specific immune responses at various time points after inoculation and viral loads following challenge 35 days postvaccination. (B) To measure antibody titers, BALB/c mice (n = 5 per group) were inoculated via the intranasal, subcutaneous, or intramuscular route with split virus vaccine alone or with split virus combined with R4Pam2Cys. Sera were obtained from blood taken 34 days later, and vaccine-specific antibody levels were determined by ELISA using split virus vaccine as a coating antigen. (C) Isotypes of vaccine-specific antibodies in sera and lung homogenate supernatants from intranasally vaccinated mice were also assayed using isotype-specific detection antibodies. Results are presented as the mean antibody titer (± SD) from all serum samples in each group. (D) To analyze CD8+ T cell responses, BALB/c mice (n = 3 per group) were inoculated via the intranasal route and lymphocytes in the lungs and MLNs were stained with fluorochrome-conjugated anti-CD8 antibody and H2Kd tetramer to detect NP147–155-specific cells. (E) Lymphocytes were also restimulated in vitro in the presence of NP147–155 for 7 days before analysis for IFN-γ-, TNF-α-, and IL-2-producing CD8+ T cells by ICS. Bar graphs depict the means numbers of NP147–155 tetramer+-producing (D) and antigen-specific cytokine-producing (E) CD8+ T cells per group, and asterisks (*) indicate P values of <0.05.
FIG 4
FIG 4
Protection of vaccinated animals against homologous and heterologous virus. BALB/c mice (n = 7 to 15 per group) were intranasally inoculated with PR8-derived split virus vaccine alone or formulated with R4Pam2Cys. A separate group of mice also received a similar dose of R4Pam2Cys alone. Animals were challenged intranasally 35 days later with 50 PFU of PR8 (A) or 104.5 PFU of X31 (B). Titers of virus in lung homogenates collected 5 days after viral challenge were determined by plaque formation. Viral titers of individual animals are presented, with the mean value of the group represented by the horizontal bar. Error bars represent SD, and asterisks (*) indicate P values of <0.05.
FIG 5
FIG 5
Cross-reactive immunity and antibody responses in vaccinated µMT mice. (A) C57BL/6 mice (white bars) or congenic µMT mice (black bars) were inoculated via the intranasal route with split virus vaccine alone or combined with R4Pam2Cys (n = 8 to 10 mice per group). Vaccine-specific antibody levels in serum and lung homogenates were measured 34 days later by ELISA. The means and SDs of the results determined for each group are shown. (B) Animals were challenged 35 days after vaccination with 104.5 PFU of X31. Titers of virus in individual lung homogenates collected from C57BL/6 mice (white circles) and µMT mice (black circles) 5 days after viral challenge were determined by plaque formation. The mean values are represented by a horizontal bar. Error bars represent SD, and asterisks (*) indicate P values of <0.05.
FIG 6
FIG 6
Induction of CD8+ T cell responses in vaccinated mice following X31 challenge and its role in mediating cross-reactivity immunity. (A) BALB/c mice (n = 5 per group) were inoculated with split virus vaccine alone or premixed with R4Pam2Cys. Lymphocytes in lungs of vaccinated mice were analyzed for virus- or NP147–155-specific IFN-γ-producing T cells in an ICS assay on day 34 postvaccination. Animals were then challenged on day 35 with 104.5 PFU of X31, and specific IFN-γ-producing T cells in the lungs were enumerated 5 days later. Each bar represents the mean and SD of the results determined for each group. (B) Concatenated dot plots of samples from virus-challenged animals are also shown, depicting the percentages (± SD) of responsive IFN-γ+ CD8+ T cells in the lung following stimulation with virus-infected APCs. (C) Vaccinated mice (n = 5 to 10) were also depleted of CD8+ T cells prior to X31 challenge. The total numbers of NP147–155-specific IFN-γ+ CD8+ T cells in BAL fluid samples from CD8+ T cell-depleted or undepleted vaccinated mice were enumerated in an ICS assay 5 days later. (D) Titers of virus in lung homogenates were also determined by plaque formation. Titers in individual animals are presented, with the mean values for the groups represented by the horizontal bars. All error bars represent SD, and asterisks (*) indicate P values of <0.05.
FIG 7
FIG 7
Pulmonary infection of unvaccinated contact mice cohoused with vaccinated index mice. BALB/c mice (n = 4 per group) were inoculated with PBS or with split virus vaccine alone or premixed with R4Pam2Cys. Mice were then challenged 35 days later with 104.5 PFU of X31. After 6 h, 2 vaccinated index mice were cohoused with 3 naive unvaccinated contact mice for 48 h in a box that allowed direct contact. Vaccinated index mice were culled at 54 h postchallenge, while contact mice were culled at 96 h after the cohousing. Lungs were collected to assess viral loads as determined by plaque assay. Averaged geometric mean viral titers (± SD) in the lungs of mice determined in two separate experiments that included 8 index mice and 12 contact mice are shown.
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