Introduction

According to the World Health Organization, the annual global incidence of influenza is about 1 billion, with up to 500,000 deaths [1]. The disease is especially severe in children. The most effective means of prophylaxis against influenza and associated complications is vaccination. But seasonal immunity cannot ensure protection in subsequent influenza seasons, mostly because of changes in strain circulation, changes in antigen drift, and decreased immunity. Influenza vaccines are updated annually to include stains predicted to circulate in the coming winter [2, 3].

Currently, different types of vaccines are used, including inactivated, live attenuated, and recombinant subunit vaccines. All of them are mostly trivalent or quadrivalent. Influenza vaccines are given intramuscularly, intracutaneously, or intranasally. The immune system responds mostly to hemagglutinin and neuraminidase, surface glycoprotein antigens of influenza virus [4]. The immunodominant epitopes of these antigens differ greatly between viral strains. For this reason, vaccines protect only against strains included in them.

These limitations have spurred interest in recombinant proteins based on viral antigens. Recombinant vaccines have advantages over live or inactivated ones, because they are usually well purified and characterized; therefore, they are safer and are better suited for large-scale production. Recombinant vaccines also have some drawbacks. For example, an antigen per se is usually weakly immunogenic and requires the use of adjuvants and/or delivery systems (in particular, viruslike particles) [5, 6].

A promising antigen for recombinant vaccines is the surface protein M2e (extracellular domain of Matrix 2 protein). The gene coding for the structure of this protein is preserved unchanged in all influenza A strains; consequently, all viruses can be recognized and eliminated by the immune system of vaccinated persons [7]. Anti-M2e antibodies limit virus replication and viral plaque formation in a cell monolayer in vitro and induce protection against virus subtypes within group A [8]. Anti-M2e antibodies were identified that cross-reacted with seasonal, pandemic H1N1, and highly pathogenic avian H5N1 strains [9]. Although anti-M2 antibodies do not neutralize viruses, they ensure antibody-dependent cellular cytotoxicity and are, therefore, important in the immune response to influenza virus [8]. Nonetheless, the application of M2e in vaccine design is restricted by the need for highly immunogenic carriers. There is good reason, therefore, to develop methods for increasing M2e immunogenicity and for using N2e in combination with various stimulants of cellular immune response [10, 11].

Much current interest is in the use of nanoparticles as platforms for vaccines [12,13,14,15], including virus vaccines [16,17,18]. Carriers for experimental influenza vaccines include liposomes [19], ImmunoStimulating COMplexes (ISCOMs) [20], calcium phosphate nanoparticles [21], polymer nanoparticles [22], and silica nanoparticles [23].

Among the most promising antigen carriers used in immunization are gold nanoparticles (AuNPs) [24,25,26]. This is because, besides being able to carry antigens, AuNPs also have adjuvant properties [27, 28]. AuNP uptake into immune cells activates the production of proinflammatory cytokines, a finding which indicates directly that AuNPs are immunostimulatory. The activation of immune cells by AuNPs may form a basis for the development of new vaccine adjuvants [29].

AuNPs have been used to develop prototypes of influenza vaccines and generate antibodies against influenza virus antigens. The specific antigens were hemagglutinin [30] and two matrix proteins of influenza virus, M1 [31] and M2 [32, 33] (both synthetic and recombinant).

In 2014, the team led by Harvinder Gill proposed a prototype intranasal influenza vaccine consisting of a synthetic M2e peptide conjugated to AuNPs, with CpG oligodeoxynucleotide (CpG ODN) as the adjuvant [34]. The conjugate induced specific antiviral IgG and protected mice against a lethal dose of PR8 influenza virus. Subsequently, Gill’s team reported more detailed results from the use of their prototype vaccine [35, 36].

In recent work, the antibody titer was the highest in the sera of mice immunized simultaneously with antigen–AuNPs and CpG–AuNPs conjugates [37]. Here, we examined the effect of AuNPs conjugated to a synthetic M2e peptide on immune response in intraperitoneally immunized mice.

Materials and methods

Preparation of gold nanoparticle conjugates

The antigen used for immunization was synthetic M2e peptide [acetylated-SLLTEVETPIRNEWGSRSNDSSD-amidated; molecular mass, 2736 Da (Cytokine Co., Russia)].

Spherical AuNPs (average diameter, 15 nm) were made according to Frens [38] by the reduction of tetrachloroauric acid (Sigma-Aldrich, USA) with sodium citrate (Fluka, Switzerland). A 242.5-mL portion of 0.01% aqueous tetrachloroauric acid was heated to 100 °C on a magnetic stirrer in an Erlenmeyer flask fitted with a water-cooled reflux tube. Then, 7.5 mL of 1% aqueous sodium citrate was added and the mixture was boiled for a further 30 min until a bright-red sol formed.

Particle characteristics were measured with a Libra 120 transmission electron microscope (Carl Zeiss), a Specord S 250 spectrophotometer (Analytik Jena), and a Zetasizer Nano-ZS particle size and zeta potential analyzer (Malvern). All measurements were made at the Simbioz Center for the Collective Use of Research Equipment at the Institute of Biochemistry and Physiology of Plants and Microorganisms.

To prepare antigen–15-nm AuNP conjugates, we estimated the “gold number” (minimal amount of antigen that protects the sol against salt aggregation) for the M2e solution. To this end, 20 μl of an antigen solution (initial concentration, 1 mg mL−1) was titrated twofold on a 96-well microtiter plate. Each well-received 200 μL of AuNPs (A520 = 1.0) and 20 μL of 1.7 M NaCl. The minimal stabilizing concentration for the antigen was 3 μg mL−1. Conjugation was done by simple mixing, and no coupling agents were added. A tenfold excess of the peptide was used, because an excess of a soluble antigen not only does not interfere with immunization but actually facilitates an increase in antibody production [35]. The resultant conjugates are aggregationally stable under conditions close to physiological.

AuNPs (15 nm) were conjugated to 5′-thiolated CpG ODN (Syntol) as described earlier [37, 39]: 100 μl of aqueous ODN was added to 2 ml of AuNP solution. After overnight incubation, the NaCl concentration was increased to 0.1 M with 1 M PBS (pH 7.2). The final mixture was shaken for an additional 24 h, centrifuged at 15000g for 20 min, and resuspended in 0.01 M PBS, containing 0.1 M NaCl.

Animal immunization

For immunization, BALB/c white mice were divided into six groups of six in each, and they received injections as follows:

  1. 1.

    “Grippol” vaccine (Microgen, Russia)

  2. 2.

    М2е + CFA (complete Freund’s adjuvant; Sigma-Aldrich, USA)

  3. 3.

    М2е + AuNPs

  4. 4.

    М2е + AuNPs + CpG + AuNPs

  5. 5.

    AuNPs

  6. 6.

    PBS

The dose of M2e in groups 2 to 4 was constant (15 μg). The dose of protein antigens in group 1 was ~ 15 μg. The animals were immunized intraperitoneally by two 50-μL injections with an interval of 10 days in-between. Besides AuNPs, other adjuvants used were CFA and AuNP-conjugated CpG ODN 1826. The animals were killed, and sera were collected on day 42 of the experiment for measurements of antibody titers and interleukin concentrations. In parallel, we measured the respiratory activity of splenic lymphocytes and peritoneal macrophages.

Animal care complied with the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes, the Guide for the Care and Use of Laboratory Animals, and Russian legislation.

Immunological and toxicological analysis

Peritoneal macrophages and splenic lymphocytes were isolated as described earlier [40]. Antibody titers were estimated by enzyme-linked immunosorbent assay (ELISA) with horseradish peroxidase-labeled antibodies against mouse IgG (Jackson ImmunoResearch, UK). The synthetic peptide was used as the immobilized antigen. The reaction results were obtained on a Multiskan Ascent microplate spectrophotometer (Thermo, USA), as described earlier [37].

To measure the serum interleukin concentration, we used a Plate Screen analyzer (Hospitex Diagnostics, Italy) and reagent kits for IL-1β, IL-6, and IFN-γ (Vector-Best, Russia).

Respiratory activity was measured by the ability of immune cells to reduce nitrotetrazolium blue (MTT; Sigma-Aldrich) to formazan [41]. The concentration of reduced formazan was converted to that per cell [40].

Statistics

Data were statistically processed with Excel 2007 software (Microsoft Corp., USA). The standard error of the mean and its confidence limits were calculated by Student’s t test (p = 0.05). The significance of between-samples differences was estimated by a two-sample unpaired Student’s t test with unequal variances. Differences were considered significant at p ≤ 0.05.

Results and discussion

The synthesized Au nanospheres were characterized by transmission electron microscopy, spectrophotometry, and dynamic light scattering. The measured data for the 15-nm particles were as follows: average diameter, 15.2 nm; λmax, 517.1 nm; A520, 1.1; and the number of particles per ml, 1.6 × 1012. The Au concentration was 57 μg ml−1.

Mice were immunized with “Grippol” commercial influenza vaccine and with the M2e antigen in combination with different adjuvants. Antibody titers were estimated by ELISA and are expressed in the ordinary way and as log2 (Table 1). The highest titer was obtained from the simultaneous use of two adjuvants—AuNPs and AuNPs + CpG (1:12800). This titer was about threefold greater than the titers obtained with AuNPs and CFA and was about sevenfold greater than the titer obtained with “Grippol” vaccine.

Table 1 Antibody titers obtained with different adjuvants
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Therefore, the production of antibodies can be increased when the animals are immunized simultaneously with М2е + AuNPs and CpG + AuNPs.

The respiratory activity of peritoneal macrophages and splenic lymphocytes was measured by the MTT assay. The concentration of reduced formazan was converted to that per cell (Table 2). The respiratory activity of peritoneal macrophages was highest in the animals given М2е + AuNPs + CpG + AuNPs, increasing by about 40% (р = 0.0042) relative to PBS and by about 25% (р = 0.039) relative to the commercial vaccine. М2е + AuNPs and М2е + CFA were almost equal in their effect on dehydrogenase activity in peritoneal macrophages (р = 0.73). Similar results were obtained for the splenic lymphocytes. These findings could be explained by the more effective penetration of AuNP conjugates into phagocytic cells, with improved antigen presentation to the antibody-producing cells.

Table 2 Formazan concentrations for macrophages and lymphocytes
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We also measured the production of the proinflammatory cytokines IL-1β, IL-6, and IFN-γ in sera from the immunized animals (Figs. 1, 2, and 3). The content of IFN-γ obtained with М2е + AuNPs + CpG + AuNPs was 1.5-fold greater than that obtained with М2е + AuNPs (р = 0.0022) or with M2e + CFA (р = 0.0008). It also was 5.6-fold greater than the IFN-γ content obtained with the commercial vaccine. The production of IL-1β did not differ significantly among the groups of mice. The production of IL-6 increased significantly with М2е + AuNPs, M2e + CFA, and М2е + AuNPs + CpG + AuNPs, as compared with the use of the commercial vaccine.

Fig. 1
figure 1

Concentration of IFN-γ in sera of animals immunized with M2e in combination with different adjuvants

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Fig. 2
figure 2

Concentration of IL-1β in sera of animals immunized with M2e in combination with different adjuvants

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Fig. 3
figure 3

Concentration of IL-6 in sera of animals immunized with M2e in combination with different adjuvants

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IFN-γ, which mediates inflammation in viral infections, inhibits virus replication, promotes the expression of MHC II, and activates NK cells and macrophages [42]. IL-6 helps B lymphocytes mature into antibody-producing cells [42]. Therefore, the increased concentration of IL-6 indicates that immunization with М2е + AuNPs activated the polyclonal production of Ig.

Conclusions

Thus, of the immunogens tested in this study (including a commercial vaccine), М2е + AuNPs + CpG + AuNPs was most effective, producing antibodies with the highest titer. It also was better at increasing the respiratory activity of lymphoid cells and the production of proinflammatory cytokines. The immunostimulatory (adjuvant) effect of AuNPs may be due to the more effective penetration of conjugates into phagocytic cells, which leads to improved antigen presentation to the antibody-producing cells. Consequently, the use of AuNPs as an antigen carrier leads to a complete and coordinated immune response from both cellular and humoral immunity.

AuNPs can be used as an adjuvant to improve the effectiveness of vaccines, stimulate antigen-presenting cells, and provide controlled release of antigens. In addition, the immunogenicity of AuNPs is determined by their physicochemical properties, such as size, shape, charge, and surface functionalization. Studying the immune response from the use of AuNPs as a carrier and adjuvant in antibody preparation will allow evaluation of their potential for use in vaccine design [43, 44]. Such nanotechnology would improve vaccine safety, potency, and availability, offering compelling platforms toward addressing many public health threats. More comprehensive studies of AuNPs are expected to reveal further applications. With increasing knowledge in immunology that uncovers the profound immune responses needed for effective defense, AuNPs are poised to attract growing attention in vaccine development.