A formulated poly (I:C)/CCL21 as an effective mucosal adjuvant for gamma-irradiated influenza vaccine

Appropriate inactivation techniques, which abolish viral infectious titers while preserving the immunogenicity of viruses, should be employed to develop whole-virion vaccines able to induce cross-immunity. Inducing chemical changes using formaldehyde or β-propiolactone along with incorporation of physical treatments such as ultraviolet light or gamma-rays are the prevailing procedures used for the preparation of inactivated influenza viruses [1]. However, the comparison of three different sterilization methods has shown that gamma-irradiated influenza A virus (γ-Flu) significantly contributes to T-cell cross-reactivity, due to the ability of gamma-rays to preserve most of the antigenic structure and biological integrity of proteins during treatment, making it a promising vaccine candidate to overcome the low efficacy of current Flu-vaccines against antigenic variants of influenza virus [1‐6].

More importantly, radiation conditions including gamma-irradiation dose and the temperature of radiation, -as well as the Bremsstrahlung process, the secondary radiation produced during treatment with gamma-irradiation, may introduce unwanted structural damages in viral proteins upon the pathogen inactivation [6, 7]. Recently, adverse effects of non-optimized experimental inactivation conditions, such as high radiation dose and high temperature, on the immunogenicity of the γ-Flu vaccine have been evaluated [6]. Besides, mucosal immunization by intranasal administration of inactivated vaccines without appropriate mucosal adjuvants is often not sufficiently effective [8‐10]. Therefore, regarding nasal gamma-based vaccine development, the use of proper adjuvants may provide a rational approach for increasing vaccine efficacy [9, 11]. Of note, vaccine adjuvant incorporation strategy may also be beneficial for decreasing the reactogenicity of whole inactivated Flu viruses by reducing vaccine doses required to achieve sufficient protective immunity [11, 12].

Targeting dendritic cell (DC) receptors by antigens such as polyriboinosinic-polyribocytidylic acid (poly (I:C)) and chemokine (C–C motif) ligand 21 (CCL21) has been shown to significantly improve vaccine immunization via supporting adaptive immunity, indicating their potential clinical value [10, 13‐17]. Poly (I:C) is an artificial form of viral double-stranded RNA (dsRNA), which induces the activation of toll-like receptor 3 (TLR3) and cytoplasmic dsRNA sensors (such as melanocyte differentiation-associated 5 (MDA5), retinoic acid-inducible gene I (RIG-I), and the NLR pyrin domain containing 3 (Nlrp3)), as well as the dictation of their downstream signaling pathways, resulting in cytokine production, up-regulation of co-stimulatory molecules and DCs maturation [18]. Mechanistically, the adjuvant activity of poly (I:C) is more related to its superior ability for inducing type 1 interferon (IFN-1), which is directly associated with the linkage of innate and adaptive immune responses [13]. CCL21, a ligand for CC chemokine receptor 7 (CCR7), has also been known to be vital for priming responses of adaptive immunity. Owing to the role of CCL21 as the main regulator of lymphatic migration of CCR7⁺ immune cells, notably DCs and T-helper 1 (Th1) lymphocytes to T-cell regions of secondary lymphoid organs, which is a critical step in orchestrating adaptive T-cell responses, it can be considered as an attractive adjuvant to facilitate vaccines to boost immunity [15, 19, 20]. In addition, the pre-clinical and clinical researches using different tumor models have revealed the efficacy of poly (I:C) and CCL21 for using as a cancer vaccine adjuvant, making them promising candidates for developing a reasonable and safe vaccine adjuvant incorporation strategy [15, 21, 22].

Recently, the activity of poly (I:C) as a mucosal vaccine adjuvant and adaptive-immunity activator has been successfully assessed in animal models of influenza infection [8, 10, 23‐25]. CCL21 is also the well-studied lymphoid chemokine adjuvant, and when applied in a mucosal prime/boost regimen against herpes simplex virus type-1 (HSV-1), led to further improvement of protection by the vaccine [26]. Accordingly, this study was undertaken to evaluate whether intranasal immunization of BALB/c mice with gamma-irradiated H1N1 vaccine plus poly (I:C) and recombinant murine CCL21, as adjuvants, induced improved protective immune responses against lethal influenza virus challenge.

Vaccination has been developing as the most assured strategy of reducing the problems associated with antiviral therapy in patients contending with the influenza virus, particularly of reducing medical expenses [30]. However, the efficacy of current vaccine platforms can be limited by (a) the high prevalence of antigenic variations in the influenza viruses; (b) the lack of cross-protective immunity [12, 31]; (c) poor immunogenicity among people with risk factors for infection; and (d) vaccine shortage at pandemic events due to their lengthy production time [12, 32, 33]. Accordingly, the genesis of a novel, more sophisticated generation of vaccines should be considered [12].

Preserving the most of immunostimulatory pathogen's epitopes upon gamma-inactivation, which are needed for the induction of an effective coverage against heterologous strains, the high penetration power of γ-rays into biological materials, high safety profile due to impossibility of returning γ-inactivated viruses to virulent type, low requirement for staff training, and the feasibility of rapid preparation of vaccine at large-scale, have led to increased global-attention to gamma-rays as an attractive approach for vaccine manufacturing [1, 34].

Compared to other forms of inactivation approaches, particularly chemical and UV treatments, and regarding the ability of gamma radiation to inactivate pathogens at the deeply frozen state and subsequently to reduce the oxidative alterations of protein epitopes by minimizing free radicals damage provided through water radiolysis, the antiviral activity of ionizing radiation is mainly attributed to preferential absorbance of the gamma photons by nucleic acids, leading to loss of genome integrity. Therefore, efficient cellular uptake and un-coating of inactivated Flu viruses is the most probable mechanism by which gamma radiation can induce cross-reactive cytotoxic T- lymphocyte (CTL) responses [5, 34]. Recent studies in mice with gamma-inactivated influenza whole-virion vaccine have revealed its superior efficacy in improving antibody-mediated immunity and inducing the high-level of cellular immune responses, as well as in conferring the homologous or heterologous immunity [1‐3, 6]. We report here that the mouse-adapted human influenza virus strain A/PR/8/34 [A/Puerto Rico/8/34 (H1N1)], rendered non-infectious by exposure to 28 kGy of gamma-irradiation, can: (I) induce significant virus-specific humoral, mucosal, and cell-mediated immunity in mice; (II) modulate the lung inflammatory responses; and most importantly (III) protect against lethal infection by homotypic influenza viruses, which is consistent with previously published work.

It is important to note that due to the lack of precise optimization of radiation conditions coupled with the Bremsstrahlung process, there is typically the possibility of introducing unwanted alterations in vaccine epitopes, leading to reduction in the effectiveness of the gamma-inactivated vaccine [6, 7]. Furthermore, upon the early attempts for the annual influenza vaccination and due to the whole inactivated influenza vaccine adverse events, safer vaccines (i.e., the split virion influenza vaccine) were developed [12]. Besides, nasal vaccines, notably those that influence the quality and quantity of the adaptive immunity and subsequently induce a robust protective T-cell immune response, are composed of antigens and adjuvants [8‐10]. Thus, it seems that the nasal gamma-based inactivated Flu vaccines require adjuvants to reduce their reactogenicity, as well as to improve their immunogenicity. We have previously discussed the superiority of plasmid encoding mouse interleukin-28B (mIL-28B) as a mucosal adjuvant in terms of improved immunity to the whole gamma-irradiated influenza A (subtype H1N1) vaccine [11]. However, there is an increasing demand for safer and more effective vaccine formulations to decline annual influenza fatality rate.

Double-stranded RNA analog Poly (I:C) contributes to the up-regulation of multiple signaling pathways, which leads to increase in DC presentation capacity and in DC migration to the lymphatic organ where the adaptive immunity is shaped [13]. The influence of intranasal administration of Poly (I:C) as a mucosal adjuvant with the vaccine candidate to facilitate the elicitation of a strong adaptive immunity has been well documented [8, 23, 24]. Ichinohe et al. demonstrated that intranasal co-delivery of poly (I:C) as an adjuvant could elevate the level of alpha/beta interferons, as well as Th1- and Th2-associated cytokines induced by the inactivated influenza virus HA vaccine [14]. Another study conducted by Zang et al. revealed the efficacy of poly (I:C) as a mucosal adjuvant in elevating both humoral and cellular immune responses to inactivated H9N2 vaccine when included in the vaccination regimen [10]. On the other hand, poly (I:C), administered intranasally in aged mice, has recently been shown to induce the protection against lethal respiratory virus infections, without any side effects, by up-regulating the expression of IFN-β, IFN-γ, IL-1β, and tumor necrosis factor (TNF) in the lungs, indicating the potential applicability of poly (I:C) as a prophylactic agent in aged individuals with risk factors for severe acute respiratory syndrome coronavirus (SARS-CoV) or influenza A virus (IAV) infections [35].

The immunotherapeutic capability of CCL21, as a CCR7 ligand, has also been underscored by its ability to induce the CCR7-dependent recruitment of immunocytes into the secondary lymphoid tissues, resulting in the development of adaptive immunity (notably T-cell proliferation) [15, 19, 20]. The efficacy of CCL21 as a mucosal adjuvant to enhance the specific types of immunity was evaluated by Toka and colleagues. The results showed that intranasal vaccination of mice with a plasmid DNA or recombinant vaccinia virus encoding herpes simplex virus gB accompanied by the CCL21 adjuvant greatly improved the production of IgG and IgA in the serum and vaginal samples, respectively, and highly increased cytotoxic T lymphocyte responses. Furthermore, due to a rapid recall response, CCL21 adjuvanted vaccine provided the significant protection against the viral challenge [26]. Hence, the objective of the present study was to apply the murine recombinant CCL21 and poly (I:C), either alone or in combination with each other, as mucosal adjuvants to augment the immunogenicity of the gamma-irradiated H1N1 vaccine.

The results illustrated that compared to the γ-Flu alone immunized group, intranasal co-administration of CCL21or poly (I:C) individually with the γ-Flu vaccine induced a strong specific immune response, as evidenced by increasing the levels of Th1-type cytokines including IFN-γ and IL-12 compared to the concentration of IL-4 as the Th2 indicator, along with an increase in the ratio of the Th1-associated IgG2a to the Th2-associated IgG1 subtype. Increased serum IgG responses to vaccine candidate, as well as a higher trend toward the proliferative response and the release of granzyme B in the spleen of mice treated with the γ-Flu plus CCL21 or poly (I:C), as compared with those receiving γ-Flu alone, was correlated with more effective protection after a lethal influenza virus challenge (10 LD50). Furthermore, both CCL21 and Poly (I:C) adjuvants could prompt higher rates of IgA response in BAL samples compared with the non-adjuvanted vaccine. Nonetheless, based on the ELISA data, the level of antigen-specific mucosal IgA antibody in the nose was similar between the mice received the γ-Flu alone or with each adjuvant separately. However, our results also showed that the IgA response in the upper (nasal washes) and the lower (BAL fluids) respiratory tract was significantly higher than those primed with CCL21, poly (I:C), and PBS. It should be noted that mucosal immunity represents the host's first line of defense against influenza virus, which is involved in preventing the spread of infectious particles by IgA-mediated virus neutralization [36, 37]. Upper and lower respiratory tract sampling to assess the mucosal IgA concentrations showed that the level of IgA in the BAL fluids was higher than IgA titers in nasal washes. This finding is consistent with a study conducted by Song and his group [38] and our previously published work [11], reinforcing the key role of the presence of antigen-specific neutralizing antibodies in the lower respiratory tract in preventing influenza virus infection. Taken together, the current study highlighted the adjuvanticity of CCL21 or poly (I:C) to increase the immunogenicity of the γ-Flu vaccine, when co-delivered to the mucosal sites.

It is important to note that upon combining CCL21 with poly (I:C), a more potent cell- and antibody-mediated immunity in response to the γ-Flu vaccine was elicited, resulting in 100% survival following a high lethal dose challenge of A/PR/8/34 (H1N1) influenza virus. The CCL21 and poly (I:C) combination adjuvant was also able to strongly promote Th1-oriented responses with a higher ratio of IgG2a/Th1 to IgG1/Th2, as well as with an increased Th1-related cytokines (IFN-γ and IL-12). A comparison of the three adjuvant combinations revealed that CCL21 in conjugation with poly (I:C) effectively improved the mucosal antibody response in the BALF and nasal samples, as well as significantly promoted the vaccine-induced cytotoxic responses by increasing the levels of granzyme B and the CD4⁺ T-cell proliferation. Consequently, intranasal treatment of mice with CCL21 and Poly (I:C) combination adjuvant was more effective than any adjuvant alone, indicating the synergistic effects of CCL21 and poly (I:C) on the vaccine-induced immune responses.

Next, we analyzed the level of IL-6- and IL-10 cytokines in the lung homogenates of immunized mice to assess the effect of CCL21 and poly (I:C), either alone or in combination with each other, as mucosal adjuvants on the modulation of inflammatory responses. New data highlight the correlation between the excessive production of IL-6, as a major inflammatory mediator, and the hyperinflammatory state during influenza infection and subsequent increase in the illness severity [39‐41]. IL-10 is also essential for dampening the magnitude of antigen-specific immune responses to reduce host damage by negatively regulating innate and adaptive immunity. However, the exact role of IL-10 in influenza pathogenesis remains unclear and controversial results (detrimental or protective roles) for IL-10 upon viral infection have been reported [42‐44]. Recent studies in mice have shown that IL-10 levels are inversely related to the severity of influenza A virus-induced disease. Sung and his group revealed the adverse effect of IL-10 on protective pulmonary antibody immunity by interfering with the initial CD4⁺ T-cell function. A detrimental effect of IL-10 on the development of Th17-type immune responses and subsequent reduction in survival rate following a lethal challenge with a high dose of influenza has also been reported recently [42, 43]. Our findings demonstrate that the γ-Flu vaccine alone or combined with CCL21, poly (I:C), or CCL21-poly (I:C) efficiently reduced IL-6 concentrations, but differences were not significant. A similar trend was also observed for IL-10 in lung homogenates, with less IL-10 secretion obtained from immunized mice with the γ-Flu alone or with each adjuvant separately. However, the ability of CCL21 and poly (I:C) combination adjuvant in conjunction with the γ-Flu vaccine to attenuate the production of IL-10 is highly desirable, which provides further support for the concomitant administration of CCL21 and poly (I:C) as a potent mucosal adjuvant.

The current study illustrates the importance of incorporating CCL21 and Poly (I:C) combination adjuvant in the γ-Flu vaccine formulation for improving vaccine-mediated protection against intranasal challenge with 10 LD50 of H1N1 influenza viruses by increasing the ratio of IgG2a, IFN- γ, and IL-12 as Th1 indicators and the level of IgA- and IgG-manifested humoral immunity, as well as by increasing lymphocytic proliferation and cytotoxic activity of spleen cells. Furthermore, the CCL21 and Poly (I:C) combination adjuvant has a potent propensity to reduce inflammatory cytokines, notably IL-10, indicating the synergistic effects of two different types of immunological adjuvants on vaccine responses.