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. Author manuscript; available in PMC: 2023 May 18.
Published in final edited form as: Vaccine. 2020 Jun 12;38(33):5256–5267. doi: 10.1016/j.vaccine.2020.05.085

Vaccination of aged mice with adjuvanted recombinant influenza nucleoprotein enhances protective immunity

Tres Cookenham a,1, Kathleen G Lanzer a,1, Emily Gage b, Erica C Lorenzo c, Darrick Carter b,2, Rhea N Coler b,3, Susan L Baldwin b,4, Laura Haynes c, William W Reiley a, Marcia A Blackman a,*
PMCID: PMC10193286  NIHMSID: NIHMS1895790  PMID: 32540272

Abstract

Elderly individuals are highly susceptible to influenza virus (IAV) infection and respond poorly to influenza vaccines. Although the generally accepted correlate of protection following influenza vaccination is neutralizing antibody titers, cytotoxic T cell activity has been found to be a better correlate in the elderly. This suggests that vaccines designed to protect against influenza in the elderly should induce both humoral and cellular immunity. The co-induction of T cell immunity is additionally advantageous, as virus-specific T cells are frequently cross-reactive against different strains of IAV. Here, we tested the capacity of a synthetic TLR-4 adjuvant, SLA-SE (second-generation lipid adjuvant formulated in a squalene-based oil-in-water emulsion) to elicit T cell immunity to a recombinant influenza nucleoprotein (rNP), in both young and aged mice. IAV challenge of vaccinated mice resulted in a modest increase in the numbers of NP-specific CD4 and CD8 effector T cells in the spleen, but did not increase numbers of memory phenotype CD8 T cells generated following viral clearance (compared to control vaccinated mice). Cytotoxic activity of CD8, but not CD4 T cells was increased. In addition, SLA-SE adjuvanted vaccination specifically enhanced the production of NP-specific IgG2c antibodies in both young and aged mice. Although NP-specific antibodies are not neutralizing, they can cooperate with CD8 T cells and antigen-presenting cells to enhance protective immunity. Importantly, SLA-SE adjuvanted rNP-vaccination of aged mice resulted in significantly enhanced viral clearance. In addition, vaccination of aged mice resulted in enhanced survival after lethal challenge compared to control vaccination, that approached statistical significance. These data demonstrate the potential of SLA-SE adjuvanted rNP vaccines to (i) generate both cellular and humoral immunity to relatively conserved IAV proteins and (ii) elicit protective immunity to IAV in aged mice.

Keywords: Influenza, Aging, Mouse, Adjuvant, Vaccination, Nucleoprotein

1. Introduction

Influenza virus (IAV) infection is a major cause of morbidity and mortality in the elderly. While annual influenza vaccines are strongly recommended for the elderly, responsiveness to current seasonal split virus vaccines generally decreases with increasing age [1]. Strategies to enhance vaccine-mediated protection in older individuals include increasing the dose, adding adjuvants [2,3], and utilizing recombinant antigens [4]. The correlate of protection against IAV infection is generally considered to be the titer of virus-neutralizing antibodies. However, McElhaney and colleagues have shown that the number of IAV-specific T cells is a better correlate of protection in elderly humans [5,6]. T cell immunity to IAV is desirable because T cells are able to recognize epitopes of internal, relatively invariable proteins, such as nucleoprotein (NP), resulting in responsiveness to different strains of IAV.

Glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE) is a synthetic Toll-like receptor-4 (TLR-4) agonist that has proven to be an effective adjuvant in vaccines against a variety of infections, including malaria [7] and Leishmanasis [8]. In addition, administration of seasonal split virus influenza vaccines with GLA-SE has been shown to enhance protection in elderly humans, in young and aged mice and in non-human primates [9-11]. More recently, an optimized TLR4 adjuvant, SLA-SE (second-generation lipid adjuvant formulated in a squalene-based oil in water emulsion) has been developed [12]. SLA-SE has shown strong vaccination efficacy against several pathogens, including Plasmodium falciparum [13], West Nile virus [14,15], Chlamydia trachomatis [16] and Enterotoxigenic E. coli [17].

In the current studies, we compared the efficacy of vaccines formulated with recombinant influenza nucleoprotein (rNP) and either SLA-SE or Alhydrogel. Vaccinated mice were challenged with a lethal dose of IAV and cellular and humoral immunity, viral clearance, and survival were subsequently assessed.

2. Materials and Methods

Mice.

Young female C57BL/6 mice, purchased from the Jackson Laboratories (Bar Harbor, ME, USA), were housed and aged to 18 months, under specific pathogen-free conditions at the Trudeau Institute. In some cases, aged female C57BL/6 mice (18–21 months) were acquired from the aged rodent colony of the National Institute of Aging, (National Institute of Health, Bethesda, MD, USA). Experiments were carried out under biosafety level 2 (BSL2) containment and all experiments were approved by the Institutional Animal Care and Use Committee of the Trudeau Institute.

Immunization and viral challenge.

Recombinant influenza nucleoprotein (rNP) was generated from Influenza A/PR8/34 virus as a C-terminal histidine-tagged protein in E. coli and isolated using the ProBond system (Invitrogen), as previously described [18]. SLA formulated in 2% stable emulsion (SE) (2 μg/ml) was mixed with 10 μg rNP. A small and uniform particle size over time defines a stable emulsion. The emulsion dose is referred to as the % (vol/vol) oil in the final vaccine formulation; the ratio of oil to emulsifier is kept constant. The stability of the emulsion used here had been tested by particle size using dynamic light scattering, as described previously [19]. Mice were immunized intramuscularly (i.m.) in the caudal thigh muscle, in a prime/boost/boost protocol (three weeks apart) with rNP + SLA-SE. The optimal vaccination regimen was determined by analysis of NP-specific antibodies (Supplemental Fig. 1) before prime, after a boost, and after a second boost of vaccine. Alhydrogel (Brenntag) or SE (alone) combined with rNP served as controls in different experiments. Non-immunized control mice received PBS i.m. Anesthetized (2,2,2,-tribromoethanol) mice were challenged intranasally three weeks after vaccination with 1000 EID50 A/Puerto Rico/8/34 (PR8, H1N1). Survival, weight and clinical signs were assessed daily, over 21 consecutive days. Animals exhibiting significant weight loss or that were moribund were euthanized for humane reasons.

Measurement of virus titers.

Viral titers were determined on days 5, 10 and 14 after challenge, using an MDCK assay. Whole-lung tissue was harvested in 1 mL PBS at the indicated times, homogenized, and stored at − 70 °C. Virus titers were measured using a standard viral foci assay by infecting MDCK cell monolayers with serial 5-fold dilutions of lung suspension in duplicate. 24 h after infection, monolayers were extensively washed and fixed with 80% acetone in water. Infected cell clusters were detected with a biotin-labeled mouse anti-influenza A monoclonal antibody (Millipore), followed by staining with streptavidin-AP and visualized with Fast BCIP/NBT substrate (Sigma-Aldrich). The number of viral foci units (VFU) were counted, and the data were shown as the VFU/lung (mean ± SD).

Flow cytometry.

Mice were sacrificed at the indicated times and tissues were harvested. Single-cell suspensions were prepared from spleens by passage through cell strainers. Spleen cells were depleted of erythrocytes by treatment with buffered ammonium chloride solution. Bronchoalveolar lavage (BAL) cells were collected by lavage of the lungs 5 times with 1 mL of HBSS without calcium and magnesium. Lung tissue was prepared by coarsely chopping the tissue followed by incubation in a 0.5 mg/mL solution of collagenase D (Roche) and DNase (Sigma Aldrich) for 30–45 min at 37 °C. Lymphocytes were enriched from digested lung tissue by differential centrifugation, using a gradient of 40/80% Percoll (GE Healthcare). Single-cell suspensions were incubated with Fc-block (anti-CD16/32) for 15 min on ice, followed by staining with IAV-specific MHC class I and II tetramers, NP366-374/Db and NP311-324/IAb, at room temperature for 45 min. Cells were then washed and stained with antibodies to CD8, CD44, CD69, and CD127 (eBioscience); CD8, CD27, CD62L, and CD69 (BD); CD43 (1B11), CD62L, CD4 (BioLegend); and KLRG1 (SouthernBiotech). Samples were analyzed on FACS Canto II, or LSR II flow cytometers (BD) and data were analyzed with FlowJo software. Tetramers specific for IAV were generated by the Trudeau Institute Molecular Biology Core Facility.

In vivo cytotoxicity assays.

Splenocytes from congenic (CD45.1) uninfected mice were used for targets in the CTL assay. In brief, splenocytes were divided into 4 groups and pulsed for 5 h at 37 °C with either 10 μg/ml of NP366-374/Db (MHC Class I), or NP311-324/IAb (MHC Class II), or irrelevant control peptides ORF61524-531 (MHC Class I) or M2124-138 (MHC class II). Each peptide-pulsed group of splenocytes was then labeled with a different concentration of carboxyfluorescein diacetate succinimidyl ester (CFSE). Cells pulsed with ORF61524-531 were left unlabeled. Peptide pulsed and CFSE labeled cells were then mixed at a 1:1:1:1 ratio with 1x106 cells intravenously injected into naïve mice or surviving mice that had been previously challenged with 1000 EID50 A/Puerto Rico/8/34. Spleen and lungs were then harvested from mice injected with the target splenocytes 16 h post-injection. CD45.1 donor cells were identified and enumerated using flow cytometry. The % specific lysis was calculated using the following formula: [1-(non-transferred control ratio/experimental ratio)] × 100, where “ratio” refers to the percentage of irrelevant peptide-pulsed cells: percentage of relevant peptide-pulsed cells, as described [20,21]. By convention, negative percentages are plotted as zero.

Antibody ELISAs.

Immune serum was obtained after vaccination prior to challenge and, in separate experiments, 5 and 10 days after challenge for determination of NP-specific IgG, IgG1 and IgG2c antibodies. PolySorp plates (Nunc, Rochester, NY) were coated with 1 μg/ml rNP in 0.1 M bicarbonate buffer, blocked overnight with 0.1% Tween 20 / 1% BSA. Plates were washed and developed using SureBlue tetramethylbenzidine (TMB) substrate (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD). The enzymatic reaction was stopped with 1 N H2SO4 and plates were read at 450 nm wavelength.

Luminex assays.

Bronchoalveolar lavage (BAL) was obtained from surviving mice 5 and 10 days after vaccinated mice were challenged with 1000 EID50 A/PR/8/34 virus and assessed for cytokines by a Luminex Procarta cytokine assay kit (Panomics, Inc., Fremont, CA) following the protocol provided by the manufacturer.

Albumin Assay.

BAL samples were taken on day 5 and 10 post-challenge and were subsequently assayed for albumin concentration using the commercial Albumin (BCG) Assay Kit (Colorimetric; Catalog # K554-100; BioVision Milpitas, CA), following the protocol provided by the manufacturer.

Statistical analysis.

Statistical analysis was performed using Prism 5 and Prism 8.2.1 (GraphPad Software). P-values<0.05 were considered significant.

3. Results

3.1. Vaccination strategy

To test the efficacy of the SLA-SE adjuvant, we intramuscularly vaccinated young and aged C57BL/6 mice with rNP + SLA-SE, and measured immune function following challenge with a lethal dose (1000 EID50) of influenza A/PR8/34 (PR8) (Fig. 1). Preliminary studies indicated that a prime/boost/boost protocol was optimal (Supplemental Fig. 1).

Fig. 1. Vaccination strategy.

Fig. 1.

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Young and aged female mice were vaccinated intramuscularly in a prime/boost/boost protocol with rNP and SLA-SE or control adjuvant (Alhydrogel or SE). Unvaccinated mice received PBS only. Experimental details are described in the Materials and Methods and individual Figure legends.

3.2. rNP + SLA-SE vaccination enhances survival after lethal challenge of aged mice

First generation GLA-SE-adjuvanted IAV seasonal vaccines are reported to enhance the survival of young and aged mice and NHP following challenge with IAV [10,11]. Furthermore, a GLA-SE adjuvanted recombinant hemagglutinin (rHA)-based vaccine also enhanced survival in experimental models [20].

To test the efficacy of the second generation SLA-SE adjuvant, we administered rNP + SLA-SE to young or aged C57BL/6 mice and assessed weight loss (Fig. 2A, B) and survival (Fig. 2C, D) following lethal challenge with IAV. Analysis of the weight loss data show that the aged mice generally had greater weight loss than young mice. However, there was no clear correlation between vaccination and protection from weight loss in young or aged mice. Unexpectedly, the aged mice vaccinated with rNP + Alhydrogel had earlier and more severe weight loss. However, vaccination had a clear impact on survival. As was reported with the GLA-SE adjuvanted H1N1 split IAV vaccine [11], vaccination with SLA-SE, and also Alhydrogel as a control adjuvant, significantly enhanced the survival in young mice after challenge (P < 0.001). Furthermore, vaccination with rNP + SLA-SE modestly prolonged survival in aged mice, compared to rNP + Alhydrogel, with the protection approaching statistical significance (P = 0.065). Although P = 0.065 is generally not considered significant, these data indicate that the log rank survival test of the null hypothesis which tests whether the rNP + SLA-SE and rNP + Alhydrogel treatments were equivalent yielded a low probability of 0.065. This low probability supports the conclusion that the groups differed in survival and that rNP + SLA-SE vaccination enhanced survival in aged mice.

Fig. 2. Vaccination enhances survival of young mice after lethal influenza challenge.

Fig. 2.

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Vaccinated young (Panels A, C) and aged (panels B, D) mice were challenged three weeks after the last dose of vaccine with a lethal dose of 1000 EID50 A/PR8 influenza virus. Mice were monitored daily for weight (Panels A and B)(data are presented as mean ± SEM) and clinical signs for three weeks after challenge. Moribund mice were humanely sacrificed. Survival data, (panels C, D) are representative of three independent experiments. ***, P < 0.001; **, P < 0.01. †, the P value for aged mice approached, but failed to reach significance, at P = 0.065 (Log-rank test). n = 6–10 mice per group.

3.3. rNP + SLA-SE vaccination accelerates viral clearance after lethal challenge of aged mice

We next examined the impact of rNP + SLA-SE vaccination on viral clearance following lethal challenge. Importantly, in addition to the modest enhancement of survival in aged mice (Fig. 2D), vaccination significantly accelerated viral clearance (P < 0.001) compared with unvaccinated controls (Fig. 3B). Most rNP + SLA-S E-vaccinated aged mice cleared virus by day 10, whereas only one animal each in the aged PBS and rNP + Alhydrogel groups had cleared virus at that time. By day 14, all of the aged mice vaccinated with rNP + Alhydrogel were dead. Analysis of young mice revealed a different outcome. Although we observed enhanced survival in young, vaccinated mice (Fig. 2C), there was no evidence for accelerated viral clearance in young rNP + SLA-SE-vaccinated mice compared to unvaccinated controls (Fig. 3A).

Fig. 3. Vaccination accelerates viral clearance in aged mice.

Fig. 3.

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Young (panel A) and aged (panel B) vaccinated mice were challenged with a lethal dose of 1000 EID50 A/PR8 influenza virus three weeks after the last dose of vaccine. Viral titers in surviving mice were determined at 5, 10 and 14 days post-challenge. Extra mice were set up to ensure adequate numbers of surviving mice. Data are pooled from three independent experiments and presented as individual data points with bars representing mean ± standard deviation (SD). ***, P < 0.001 (Mann Whitney). Young mice vaccinated with rNP + Alhydrogel were only assayed at day 14. All the aged mice vaccinated with rNP + Alhydrogel were dead at 14 days post-infection. n = 3–17 mice per group.

3.4. Enhancement of NP-specific CD8 T cell numbers and cytolytic function but not numbers of long-term memory cells by vaccination of aged mice with rNP + SLA-SE

Because our vaccination strategy used a recombinant influenza NP protein that has previously been shown to elicit NP-specific T cells [21,22], we measured numbers of NP366-374/Db-specific CD8 T cells in the spleen, lung and BAL following IAV challenge of vaccinated mice (Fig. 4). Tetramer staining revealed that IAV infection of both rNP + SLA-SE and rNP + Alhydrogel vaccinated aged mice resulted in increased frequencies of NP-specific CD8 T cells in the spleen and lung, and in rNP + SLA-SE-vaccinated mice in the BAL, at day 10 post-infection (compared to PBS-vaccinated control aged mice). However, this only resulted in increases in the absolute numbers of CD8 NP-specific T cells in the spleen.

Fig. 4. Induction of NP tetramer-positive CD8 T cells after challenge of vaccinated young and aged mice.

Fig. 4.

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Vaccinated young and aged mice were challenged three weeks after the last dose of vaccine with a lethal dose of 1000 EID50 A/PR8 influenza virus and analyzed 10 days post-challenge for NP-tetramer positive CD8 T cells using an NP366-374/Db tetramer. The percent of NP-tetramer positive CD8 T cells among total CD8 T cells in spleen, lung and BAL (Panel A) and absolute numbers of NP-positive CD8 T cells in the spleen, lung and BAL (Panel B) were determined. Data are pooled from two independent experiments for lung and three independent experiments for BAL and spleen, and are presented as individual data points with bars representing mean ± SD. **** P < 0.0001; ***P < 0.001; **P < 0.005; *P < 0.05 (two tailed Student’s t-test). n = 2–15 mice per group.

We next examined in vivo cytotoxic function by CD8 T cells in vaccinated or unvaccinated (PBS) young and aged mice, following challenge. Clear separation of specific and non-specific MHC class I and MHC class II CFSE-labelled targets are shown in Fig. 5A. In these studies, the control adjuvant was SE (stable emulsion). Although SE alone has adjuvant activity, it requires a higher concentration of antigen to be effective [20]. The high CTL activity in the spleens of young mice (Fig. 5B) indicates that infection induces a strong response in young mice, even in the absence of vaccination. However, vaccination was essential to obtain a good CD8 CTL response in aged mice. It can be seen that both SE- and SLA-SE-adjuvanted rNP induced CD8 CTL activity in the spleens and lungs of aged mice on day 10 post-challenge.

Fig. 5. T cell cytotoxicity induced by challenge of vaccinated young and aged mice.

Fig. 5.

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Young and aged mice were unvaccinated (PBS) or vaccinated with rNP + SE or rNP + SLA-SE. Mice were challenged three weeks after the last dose of vaccine with a lethal dose of 1000 EID50 A/PR8 influenza virus. Cytotoxic activity in surviving mice was assayed in vivo 10 days after challenge. Panel A. FACS plot showing CD45.1 CFSE-labeled targets, pulsed with either 10 μg/ml of NP366-374/Db (MHC Class I), NP311-324/IAb (MHC Class II), or irrelevant control peptides ORF61524-531 (MHC Class I) or M2124-138 (MHC class II). Panels B and C. Cytotoxic activity of CD8 T cells (panel B) and CD4 T cells (panel C) in the spleen and lung 10 days after challenge of vaccinated mice. Data are representative of two independent experiments and presented as individual data points with bars representing mean ± SD. n = 2–3 mice per group.

Effective T cell vaccines depend on the generation of memory CD8 T cells that are poised to respond quickly to secondary antigen challenge. This recall efficacy depends on the quantity, phenotype, function, and longevity of the memory T cells generated. Two phenotypic markers that have been frequently used to identify memory T cells are IL-7R (CD127) and killer cell lectin-like receptor G1 (KLRG1) [23-25]. Following the resolution of infection, effector CD8 T cells develop into short-lived effector T cells (variably termed SLECs or Tsle) or memory precursor effector cells (variably termed MPECs or Tmpe). These subsets can be distinguished by levels of CD127 (SLECs/Tsle are low and MPECs/Tmpe are high) and KLRG1 (SLECs/Tsle are high and MPECs/Tmpe are low) [23-25](Fig. 6, Panel A). Double positive cells (DPEC/Tdpe) have been shown by fate mapping analysis to lose expression of KLRG1 and develop into a subset of CD127+ long-term memory cells [26]. To assess the quality of T cell memory generated following vaccination and challenge, we examined phenotypic changes in responding NP-specific CD8 T cells from young and aged mice, at two timepoints (day 10 and 42) following virus challenge (Fig. 6, Panels B and C)). Vaccination with both rNP + SLA-SE and rNP + Alhydrogel enhanced the frequency of DPECs and MPECs in young mice on day 10 post-challenge. In contrast, aged mice vaccinated with rNP + Alhydrogel had increased frequencies of MPECs, whereas vaccination with rNP + SLA-SE had increased frequencies of SLECs, on day 10 post-challenge (Fig. 6, Panel B). However, the effects of the different vaccination regimens in surviving mice were no longer apparent 42 days after challenge, suggesting that neither the numbers nor phenotype of long-term memory T cells was permanently affected by vaccination (Fig. 6, Panel C).

Fig. 6. Phenotype of NP-specific CD8 memory T cells after challenge of vaccinated young and aged mice.

Fig. 6.

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Young and aged mice were unvaccinated (PBS), or vaccinated with rNP + Alhydrogel or rNP + SLA-SE, and challenged three weeks after the last dose of vaccine with a lethal dose of 1000 EID50 A/PR8 influenza virus. NP-specific CD8 T cells from lung were analyzed for memory phenotype markers on days 10 and 42 post-challenge. Panel A. FACS plots showing the gating strategy for identification of SLEC, DPEC and MPEC cells. Panel B. Phenotype of CD8 T cells from the lung 10 days after challenge of young and aged mice. Panel C. Phenotype of CD8 T cells from the lung 42 days after challenge of vaccinated young and aged mice. Data are presented as individual data points with bars representing mean ± SD. * P < 0.05 (two-tailed Student’s t-test). n = 2–5 mice per group.

3.5. Vaccination with rNP + SLA-SE modestly enhances NP-specific CD4 T cell frequencies and numbers, but not cytolytic activity

We next analyzed the effect of vaccination on the frequency and number of NP311-325-specific CD4 T cells using the Class II tetramer. Increased frequencies and numbers of NP-specific CD4 T cells were observed in the spleens and BAL of rNP + SLA-SE vaccinated and challenged aged mice (Fig. 7A and B). CD4-mediated cytotoxic activity following challenge was enhanced in young mice comparably by rNP + Alhydrogel and rNP + SLA-SE. However, the level of CD4 cytotoxicity after challenge of aged, vaccinated mice was not increased (Fig. 5B). Although CD4 T cells have been shown to mediate cytotoxicity to IAV [27-29], CD4 T cells mediate other effector functions, such as cytokine secretion and/or help for antibody production. Therefore, we examined the effect of vaccination on the induction of cytokines and antibodies.

Fig. 7. Induction of NP tetramer-positive CD4 T cells after challenge of vaccinated young and aged mice.

Fig. 7.

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Young and aged mice were unvaccinated (PBS), or vaccinated with rNP + Alhydrogel or rNP + SLA-SE. Mice were challenged three weeks after the last dose of vaccine with a lethal dose of 1000 EID50 A/PR8 influenza virus and analyzed for NP-specific CD4 T cells using an NP311-324/IAb tetramer at day 10 post-challenge. Panel A. The frequency of NP-positive CD4 T cells among total T cells in spleen, lung and BAL Panel B. Absolute numbers of NP-positive CD4 T cells in the spleen, lung and BAL. Data are pooled from two independent experiments for lung and three independent experiments for BAL and spleen, and are presented as individual data points with bars representing mean ± SD. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 (two-tailed Student’s t-test). n = 2–17 mice per group.

3.6. Vaccination with rNP + SLA-SE fails to skew the cytokine response to a Th1 phenotype

Previous studies have associated a defined immunological signature with different adjuvants. For example, whereas alum is known to cause increased antibody titers, GLA-SE has been shown to induce antibodies, Th1 responses and a shift from IgG1 to IgG2c antibodies [31]. Th1-biased responses (high IFNγ, TNFα and IL-2 and low levels of IL-5 and IL-10) were characteristically observed following GLA-SE-adjuvanted vaccination against several pathogens, including Leishmania, m. Tuberculosis, malaria-causing Plasmodium and IAV [8,10,30,31]. Therefore, we tested the cytokine profile in the bronchoalveolar lavage of vaccinated young and aged mice at 5 and 10 days after challenge, using Luminex-based multiplex immunoassay kits. The day 5 data (Fig. 8) were analyzed with a two-way ANOVA, the two variables being “Age” and “Treatment.” The analysis showed that the only two cytokines that correlated with both age and treatment were IFNγ and IL-4. The data were also analyzed for significant effects of treatment either within or between age groups. No effect of treatment was seen for TNFα, IL-5, IL-6 or IL-17a. However, effects of treatment for both young and aged mice were seen for IL-2, and effects of treatment for young mice were seen for IFNγ, IL-4 and IL-10. Overall, the shift to a Th1 response, consisting of increased levels of IFNγ and decreased levels of IL-5, seen previously [8,10,30,31], was not observed in aged mice in our study. Day 10 data showed no statistically significant differences and are only shown to illustrate the kinetics of the cytokine response (Fig. 8).

Fig. 8. Induction of cytokines in young and aged vaccinated mice after influenza virus challenge.

Fig. 8.

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Young and aged mice were unvaccinated (PBS), or vaccinated with rNP + Alhydrogel or rNP + SLA-SE. Three weeks after the last dose of vaccine, mice were challenged with a lethal dose of 1000 EID50 A/PR8 influenza virus. Cytokines in BAL were analyzed by Luminex at 5 and 10 days after challenge. Cytokine data were analyzed statistically by 2-way ANOVA to assess main effects due to treatment and mouse age, and statistical interactions between them. Tukey’s post-tests were used to assess differences among treatments among mice of either age. Data are presented as individual data points with bars representing mean ± SD. ****P < 0.0001, **P < 0.01, *P < 0.05. n = 2–5 mice per group.

3.7. Vaccination with rNP + SLA-SE enhances IgG2c:IgG1 ratios in young and aged mice

T cell priming also affects the isotype of immunoglobulin produced [32]. Therefore, we determined whether there was a similar effect of rNP + SLA-SE vaccination on antibody levels or isotype in young and aged mice. Analysis of antibody isotype 21 days following the second boost vaccination with rNP + SLA-SE (Fig. 9A), and 5 and 10 days after IAV challenge of vaccinated mice (Fig. 9C), showed a striking and statistically significant enhancement of the IgG2c antibody response, resulting in an enhanced IgG2c/IgG1 ratio (Fig. 9B and D). The induction of an IgG2c response in both young and aged mice contrasts with a previous report that the enhancement of IgG2c antibodies was unique to young mice [11]. Despite the failure to observe a characteristic Th1 cytokine profile in Fig. 8, the induction of an IgG2c response is indicative of rNP + SLA-SE-vaccine-induced Th1 response in both young and aged mice.

Fig. 9. Vaccination enhances the production of NP-specific IgG2c antibodies, before and after challenge.

Fig. 9.

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Young and aged mice were unvaccinated (PBS), or vaccinated with rNP + Alhydrogel, rNP + SE or rNP + SLA-SE, and challenged with a lethal dose of 1000 EID50 A/PR8 influenza virus three weeks after the last dose of vaccine. Panel A. Levels of NP-specific total IgG, IgG1 and IgG2c antibodies were determined 21 days following the second boost vaccination. Panel B. The ratio of IgG2c/IgG1 NP-specific antibodies after vaccination is plotted. Panel C. Levels of total IgG, IgG1 and IgG2c NP-specific antibodies were determined 5 and 10 days after challenge of vaccinated mice. Panel D. The ratio of IgG1/IgG2c NP-specific antibodies after challenge of vaccinated mice is plotted. Data are presented as individual data points with horizontal bars representing the mean in panels A and C and median in panels B and D. Values in Panels A and B are pooled from two independent experiments. **** P < 0.0001; *** P < 0.001; **, P < 0.01; *P < 0.05 (Mann-Whitney). n = 2–10 mice per group.

3.8. rNP + SLA-SE vaccination fails to reduce the magnitude or duration of inflammation

Antibodies have been shown to mediate diverse effects during host immunity, including the suppression of inflammation [33]. There was no indication that vaccination of aged mice reduced expression of the inflammatory cytokine IL-6 (Fig. 8). In addition, quantifying albumin levels in BAL has been shown to be a good measure of inflammation and lung damage [34]. Previous studies have shown that albumin plateaus in the lung at 6–12 days post-IAV infection of young mice, whereas albumin continued to rise in aged mice, indicating on-going IAV-induced lung damage in aged mice that is not resolved. Our analysis of albumin levels in the lung at 5 and 10 days following IAV challenge of vaccinated and unvaccinated young and aged mice revealed that rNP + SLA-SE vaccination failed to reduce albumin levels (Supplemental Fig. 2). In contrast, rNP + Alhydrogel induced a statistically significant reduction in albumin levels in aged mice 10 days after IAV challenge. The reduction of albumin in the aged, Alhydrogel-adjuvanted mice contrasts with a previous report showing that rNP vaccination administered with lipopolysaccharide reduced inflammation assessed by albumin levels in the lung, in young, but not aged mice [35].

4. Discussion

In the current study, we sought to test the efficacy of recombinant influenza NP protein, administered intramuscularly with a TLR-4 adjuvant, SLA-SE, in aged C57BL/6 mice. The data show that vaccination of aged mice with rNP + SLA-SE, but not rNP + Alhydrogel, induces enhanced survival and markedly accelerated viral clearance following infection with a lethal dose of IAV. Notably, both adjuvants clearly enhanced the survival of young mice challenged with a lethal dose of IAV, whereas rNP + SLA-SE, but not rNP + Alhydrogel enhanced survival of aged mice.

We examined various parameters to determine the immunological mechanisms underlying accelerated viral clearance and enhanced survival. The most striking immunological consequence of vaccination in these studies was the shift in the ratio of IgG2c over IgG1 NP-specific antibodies (Fig. 9). Although we failed to directly demonstrate a shift to a Th1 cytokine response (Fig. 8), it is important to note that production of IgG2c isotype antibodies is considered to be a hallmark of a Th1 response [32].Vaccination resulted in higher numbers of NP-specific CD4 and CD8 T cells in the spleen (Figs. 4 and 7) and induced strong CD8 CTL activity in IAV-challenged mice (Fig. 5B). However, there was no enhancing effect on generation or maintenance of circulating memory CD8 T cells (Fig. 6), nor a strong effect of vaccination on CD4 CTL activity (Fig. 5C). In contrast to published reports with GLA-SE or SLA-SE vaccination [8,9,11,31], there was not a clear shift in the cytokine response to Th1 in aged vaccinated and challenged animals (Fig. 8).

It has been reported that GLA-SE or SLA-SE vaccination predominantly induces IgG2c, rather than IgG1, antigen-specific antibodies. For example, IAV challenge of young mice vaccinated with influenza recombinant hemagglutinin (rHA) + GLA-SE led to a predominance of IgG2c antibodies (as compared to IAV infection of naïve mice which induced IgG1 antibodies) [20]. Furthermore, split H1N1 vaccination with GLA-SE induced a profound shift in antigen-specific antibodies from IgG1 to IgG2c following challenge in young mice [11]. Enhancement of IgG2c antibodies was also seen following vaccination with a recombinant protein from Plasmodium falciparum adjuvanted with GLA- or SLA-SE [13]. Thus, our data were consistent with the results of others.

The motivation behind our study of a T cell vaccination regimen for aged mice originated with the studies of the McElhaney laboratory, showing that cell-mediated immunity, characterized by elevated levels of granzyme B and enhanced IFNγ:IL-10 ratios, is a superior correlate of protection than antibody titers in elderly humans [5,6]. Further studies by this group showed that a split-virus vaccine adjuvanted with GLA-SE induced a Th1 cytokine response and an enhanced cytolytic T cell response [9]. We have followed up on these studies by examining the protective efficacy of SLA-SE as an adjuvant for rNP vaccination targeting T cell immunity in aged mice. Although our studies showed vaccination induced enhanced frequency of CD8 T cells in aged mice, enhancement of CD4 T cell numbers and cytotoxicity was not observed and we failed to see the enhanced IFNγ:IL-10 ratio previously observed following GLA-SE adjuvanted split influenza vaccination of elderly humans [9].

An important difference in our studies compared with those of McEhlaney in humans [9], and also another study in aged mice which used SLA-SE as an adjuvant [11], is the nature of the immunizing antigen- we used rNP for vaccination to target T cell immunity rather than the more complex and immunogenic split H1N1 IAV used in the earlier studies. The enhanced effectiveness of split H1N1 + GLA-SE vaccine over rNP + SLA-SE vaccine in aged mice likely resulted from the different immunogens used, rather than differences in the adjuvants [11,12]. In the H1N1 split vaccine study in mice, vaccinated aged mice had statistically significant enhanced survival [11], whereas in our current study, enhanced survival only approached significance (Fig. 2). Interestingly, our data show accelerated viral clearance in vaccinated aged mice (Fig. 3). In the split vaccine mouse study, young mice showed accelerated viral clearance, but this parameter was not examined in aged mice [11].

Antibodies to IAV coat proteins such as hemagglutinin induced by vaccination with split-virus vaccines play an important role in viral neutralization and generating sterilizing immunity. In contrast, anti-NP antibodies, which are directed toward an internal viral protein, are not neutralizing [36]. Although non-neutralizing antibodies are unable to prevent infection, they reduce morbidity and viral load by additional effector mechanisms, involving Fc receptors [37,38], C’-dependent lysis [39,40], enhanced interferon synthesis [41,42], or enhanced dendritic cell function [43]. Although the NP antigen is thought to be expressed internally and not exposed to circulating antibodies [36,44], the NP protein can be released from infected cells as early as 2–3 days after infection and is thus available to be recognized by antibody, resulting in the formation of immune complexes [18], which can be bound to FcR-bearing phagocytic cells [45]. The protective effect of the antibody has been shown to require the presence of memory T cells [46] and cooperation with CD8 T cells and alveolar macrophages [47]. The isotype of the non-neutralizing antibodies elicited by vaccination is important because of defined differences in the affinity of binding of different isotypes to different FcγR. Importantly, IgG2c, but not IgG1 binds with high affinity to activating receptors, FcγRl and IV [48]. Recently, it was shown that isotype-switched, non-neutralizing antibodies facilitated viral antigen capture and cross-presentation by FcγR-expressing dendritic cells, resulting in sustained antigen presentation and enhanced CD8 T cell responses [49]. Another possible mechanism involving FcR enhancement of antigen cross presentation may be mediated by the tripartite motif-containing 21 (TRIM21). TRIM21 has been shown to recognize the Fc portion of pathogen:antibody immune complexes, triggering the immune complexes for proteosomal degradation and enhanced cross presentation [50].

Previous studies have shown that the magnitude of the CD8 T cell response does not necessarily correlate with protection, because optimal NP-specific T cell-mediated protection requires cooperation with non-neutralizing antibodies [46] and alveolar macrophages [47]. This is consistent with evidence suggesting that antigen presentation and CD8 T cell activation are defective in aged mice [51,52], so mechanisms of enhancement of antigen presentation in aged mice by vaccination are important for further investigation.

An important consideration for influenza vaccination in elderly humans, is that current vaccines stimulate memory T cells, developed over a lifespan of exposure to infectious IAV and IAV vaccines. In contrast, experimental mice are housed in specific pathogen-free environments and have not been previously exposed to IAV, resulting in the absence of IAV-specific memory T cells. Thus, vaccination strategies in aged mice elicit a primary T cell response to IAV, rather than recall a pre-existing memory response. More stringent activation requirements to stimulate naïve over memory T cells may explain the absence of a CD4 cytolytic response and poorer cytokine production, although the NP-specific antibody response was robust.

Although vaccination of aged mice with rNP + SLA-SE conferred accelerated viral clearance and had modest protective effects on survival, we failed to identify the immune correlate of protection. The strongest effect on immunity was the dramatic shift in the production of IgG2c anti-NP antibodies following vaccination and challenge of vaccinated mice. It is important to continue efforts to elicit strong T cell immunity in combination with neutralizing antibodies in influenza vaccine formulations. T cells can provide broader protection than neutralizing antibodies because they target epitopes that are more highly conserved in the IAV genome. This is important not only for generating more “universal” vaccines, but also for improving the efficacy of vaccines for the elderly, where the correlate of protection has been shown to be T cell immunity [9]. Vaccines that induce high titers of neutralizing antibodies induce sterilizing immunity and prevent subsequent IAV infection, thus yielding little or no cross-reactive T cell immunity. In the absence of neutralizing antibodies for a newly emerging pandemic strain, the absence of cross-reactive T cell memory might leave the population more susceptible to a pandemic strain [53].

Supplementary Material

Supplementary data 3
Supplemental Figure 1
Supplemental Figure 2

Acknowledgements

This work was funded by the National Institutes of Health (P01AG021600 and R01AG039485), the Trudeau Institute and the European Union’s Seventh Framework Programme [FP7/2007-2013] under Grant Agreement No: 280873 ADITEC. We thank Dr. Larry Johnson for statistical analysis and Dr. David Woodland for important discussion and review of the manuscript. We are also grateful to the Trudeau Institute Animal Care Staff for their dedicated maintenance of the aged animals.

Footnotes

CRediT authorship contribution statement

Tres Cookenham: Investigation, Methodology, Formal analysis, Visualization. Kathleen G. Lanzer: Investigation, Methodology, Formal analysis, Writing - review & editing. Emily Gage: Investigation. Erica C. Lorenzo: Investigation. Darrick Carter: Conceptualization, Resources. Rhea N. Coler: Supervision. Susan L. Baldwin: Writing - review & editing, Supervision. Laura Haynes: Funding acquisition, Supervision. William W. Reiley: Writing - original draft, Funding acquisition, Supervision. Marcia A. Blackman: Conceptualization, Writing - original draft, Funding acquisition, Supervision.

Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [DC is an inventor on patents covering SLA formulations. Other authors declare no known competing financial or personal relationships that could have appeared to influence the work reported in this paper].

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.vaccine.2020.05.085.

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Supplementary Materials

Supplementary data 3
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Supplemental Figure 1
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Supplemental Figure 2
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