Squalene-adjuvanted H7N9 virus vaccine induces robust humoral immune response against H7N9 and H7N7 viruses

Recent cases of avian influenza H7N9 have caused great concerns that virus may become transmittable between humans. It is imperative to develop an effective vaccine to fight against the pandemic potential of this H7N9 influenza virus to protect human from the disease. This study aims to investigate an optimized formulation for the development of H7N9 vaccines. Various doses of H7N9 inactivated whole or split-virus antigens (0.5, 1.5, or 3 μg based on hemagglutinin content) combined with squalene-based adjuvant (AddaVAX), aluminum hydroxide Al(OH)₃ or without adjuvant were evaluated for the efficacy of H7N9 vaccine regiments in mice. With either H7N9 whole or split-virus based vaccines, AddaVAX-adjuvanted formulations were the most immunogenic in eliciting significant humoral immune response against H7N9 virus and exhibited strong cross-reactive response in hemagglutination inhibition (HAI) and viral-neutralization assays against H7N7 virus as well. In contrast, formulations with Al(OH)₃ or without adjuvant were less immunogenic and elicited lower titers of HAI and microneutralization assays against both viruses. Dose-sparing experiments suggested that the formulation with as low as 0.004 μg of split or whole virus vaccine antigens together with 50% AddaVAX provided sufficient sero-protective HAI titers and achieved essential virus-neutralizing antibody titers against H7-subtype influenza viruses in mice. Protection experiments demonstrated that the formulation of 0.004 μg to 0.5 μg of split-virion vaccines with AddaVAX conferred full protection against viral challenge up to 100 LD50 of wild-type H7N9 virus, with 0% survival in placebo group. Taken together, our study demonstrates that squalene-based adjuvant can significantly enhance the protective efficacy of H7N9 virus vaccine and provides a useful strategy to confront the potential pandemic outbreaks of H7N9 virus.     

A highly immunogenic vaccine against A/H7N9 influenza virus

Since the first case of human infection in March 2013, continued reports of H7N9 cases highlight a potential pandemic threat. Highly immunogenic vaccines to this virus are urgently needed to protect vulnerable populations who lack protective immunity. In this study, an egg- and adjuvant-independent adenoviral vector-based, hemagglutinin H7 subtype influenza vaccine (HAd-H7HA) demonstrated enhanced cell-mediated immunity as well as serum antibody responses in a mouse model. Most importantly, this vaccine provided complete protection against homologous A/H7N9 viral challenge suggesting its potential utility as a pandemic vaccine.     

Plant-derived H7 VLP vaccine elicits protective immune response against H7N9 influenza virus in mice and ferrets

In March 2013, the Chinese Centre for Disease Control and Prevention confirmed the first reported case of human infection with an avian influenza A H7N9 virus. Infection with this virus often caused severe pneumonia and acute respiratory distress syndrome resulting in a case fatality rate >35%. The risk of pandemic highlighted, once again, the need for a more rapid and scalable vaccine response capability. Here, we describe the rapid (19 days) development of a plant-derived VLP vaccine based on the hemagglutinin sequence of influenza H7N9 A/Hangzhou/1/2013. The immunogenicity of the H7 VLP vaccine was assessed in mice and ferrets after one or two intramuscular dose(s) with and without adjuvant (alum or GLA-SE™). In ferrets, we also measured H7-specific cell-mediated immunity. The mice and ferrets were then challenged with H7N9 A/Anhui/1/2013 influenza virus. A single immunization with the adjuvanted vaccine elicited a strong humoral response and protected mice against an otherwise lethal challenge. Two doses of unadjuvanted vaccine significantly increased humoral response and resulted in 100% protection with significant reduction of clinical signs leading to nearly asymptomatic infections. In ferrets, a single immunization with the alum-adjuvanted H7 VLP vaccine induced strong humoral and CMI responses with antigen-specific activation of CD3⁺ T cells. Compared to animals injected with placebo, ferrets vaccinated with alum-adjuvanted vaccine displayed no weight loss during the challenge. Moreover, the vaccination significantly reduced the viral load in lungs and nasal washes 3 days after the infection. This candidate plant-made H7 vaccine therefore induced protective responses after either one adjuvanted or two unadjuvanted doses. Studies are currently ongoing to better characterize the immune response elicited by the plant-derived VLP vaccines. Regardless, these data are very promising for the rapid production of an immunogenic and protective vaccine against this potentially pandemic virus.     

A single dose of whole inactivated H7N9 influenza vaccine confers protection from severe disease but not infection in ferrets

The H7N9 influenza virus caused significant mortality and morbidity in infected humans during an outbreak in China in 2013 stimulating vaccine development efforts. As previous H7-based vaccines have been poorly immunogenic in humans we sought to determine the immunogenic and protective properties of an inactivated whole virus vaccine derived from a 2013 H7N9 virus in ferrets. As whole virus vaccine preparations have been shown to be more immunogenic in humans, but less likely to be used, than split or surface antigen formulations, we vaccinated ferrets with a single dose of 15, 30, or 50 μg of the vaccine and subsequently challenged with wild-type A/Anhui/1/2013 (H7N9) either by direct instillation or by contact with infected animals. Although ferrets vaccinated with higher doses of vaccine had higher serum hemagglutinin inhibition (HI) titers, the titers were still low. During subsequent instillation challenge, however, ferrets vaccinated with 50 μg of vaccine showed no illness and shed significantly less virus than mock vaccinated controls. All vaccinated ferrets had lower virus loads in their lungs as compared to controls. In a separate study where unvaccinated-infected ferrets were placed in the same cage with vaccinated-uninfected ferrets, vaccination did not prevent infection in the contact ferrets, although they showed a trend of lower viral load. Overall, we conclude that inactivated whole-virus H7N9 vaccine was able to reduce the severity of infection and viral load, despite the lack of hemagglutinin-inhibiting antibodies.     

A vaccine prepared from a non-pathogenic H5N1 avian influenza virus strain confers protective immunity against highly pathogenic avian influenza virus infection in cynomolgus macaques

In order to prepare for the emergence of pandemic influenza viruses, we have established an influenza virus library that contains non-pathogenic influenza A virus strains with 135 combinations of 15 hemagglutinin and 9 neuraminidase subtypes. In this study, we developed a vaccine against H5N1 highly pathogenic avian influenza (HPAI) virus infection in humans using a virus strain selected from the library. We examined its immunogenic potency using cynomolgus macaques as a primate model. Virus antigen-specific antibodies were elicited by intranasal or subcutaneous administration of inactivated whole virus particle vaccines. After challenge with an H5N1 HPAI virus isolate obtained from a Vietnamese patient, the virus was detected only on next day following inoculation in the nasal and/or tracheal swabs of vaccinated macaques that were asymptomatic. On the other hand, the viruses were isolated from nasal and tracheal swabs from non-vaccinated macaques until day 5 and day 7 after inoculation of the H5N1 HPAI virus, respectively. Although six non-vaccinated macaques developed a high body temperature, and two of them lost their appetite after HPAI virus infection, they recovered by the end of the 12-day observation period and did not show the severe symptoms that have been reported in human H5N1 virus infection cases. This demonstrates that the vaccine prepared with the non-pathogenic H5N1 virus from our influenza virus library conferred protective immunity against H5N1 HPAI virus infection to macaques.     

Development of a high-yield reassortant influenza vaccine virus derived from the A/Anhui/1/2013 (H7N9) strain

In April 2013, the first three fatal cases of human infection with an avian influenza A virus (H7N9) were reported in China. Because of a pandemic threat by this virus, we have commenced to develop candidate vaccine viruses (CVVs). Three 6:2 genetic reassortant viruses with different hemagglutinin (HA) sequences, NIIDRG-10, -10.1 and -10.2, were generated by a reverse genetics technique between the high egg-growth master virus, A/Puerto Rico/8/34 (H1N1) and A/Anhui/1/2013 (H7N9), kindly provided by the Chinese Center for Disease Control and Prevention. The different HA gene sequences of the three CVVs were derived from the original virus stock. NIIDRG-10 possesses HA, whose sequence is identical to that of the original A/Anhui/1/2013 (H7N9) in the Global Initiative on Sharing Avian Influenza Data (EPI439507), while NIIDRG-10.1 and -10.2 possess amino acid differences, A125T and N123D/N149D, respectively, compared with NIIDRG-10. NIIDRG-10 replicated in embryonated chicken eggs with low hemagglutination titer 128, whereas NIIDRG-10.1 and -10.2 grew well with hemagglutination titer 1024. These viruses reacted well with a ferret antiserum raised against the original A/Anhui/1/2013 virus. Ferret antiserum against NIIDRG-10.1 reacted well with A/Anhui/1/2013 similar to the homologous virus NIIDRG-10.1. These results indicated that NIIDRG-10.1 passed the two-way test of antigenic identity. In contrast, the ferret antiserum against NIIDRG-10.2 reacted with A/Anhui/1/2013 at an 8-fold lower hemagglutination inhibition titer than with the homologous virus NIIDRG-10.2, indicating an antigenic change. The total and HA protein yields of NIIDRG-10.1 were 14.7 and 6.9 μg/ml, respectively, similar to those levels of high-yield seed viruses of seasonal influenza vaccines. NIIDRG-10.1 was approved as one of the CVVs for H7N9 viruses by the WHO in 2013. The candidate vaccine derived from NIIDRG-10.1 is currently being evaluated in a phase II clinical study in Japan.     

Cross-protection against H7N9 influenza strains using a live-attenuated H7N3 virus vaccine

In 2013, avian H7N9 influenza viruses were detected infecting people in China resulting in high mortality. Influenza H7 vaccines that provide cross-protection against these new viruses are needed until specific H7N9 vaccines are ready to market. In this study, an available H7N3 cold-adapted, temperature sensitive, live attenuated influenza vaccine (LAIV) elicited protective immune responses in ferrets against H7N9 viruses. The H7N3 LAIV administered alone (by intranasal or subcutaneous administration) or in a prime-boost strategy using inactivated H7N9 virus resulted in high HAI titers and protected 100% of the animals against H7N9 challenge. Naïve ferrets passively administered immune serum from H7N3 LAIV infected animals were also protected. In contrast, recombinant HA protein or inactivated viruses did not protect ferrets against challenge and elicited lower antibody titers. Thus, the H7N3 LAIV vaccine was immunogenic in healthy seronegative ferrets and protected these ferrets against the newly emerged H7N9 avian influenza virus.     

Development of Eurasian H7N7/PR8 high growth reassortant virus for clinical evaluation as an inactivated pandemic influenza vaccine

Avian-to-human transmission of the high pathogenicity (HP) H7N7 subtype avian influenza viruses in the Netherlands during 2003 caused zoonotic infections in 89 people, including a case of acute fatal respiratory distress syndrome. Public health emergency preparedness against H7N7 avian influenza viruses with pandemic potential includes the development of vaccine candidate viruses. In order to develop a high growth reassortant vaccine candidate virus, low pathogenicity (LP) A/mallard/Netherlands/12/2000 (H7N3) and A/mallard/Netherlands/2/2000 (H10N7) strains were selected as donors of the H7 haemagglutinin and N7 neuraminidase genes, respectively. The donor viruses exhibited high amino acid sequence homology with the surface glycoproteins of A/Netherlands/219/03 H7N7 virus (NL219), an isolate recovered from the fatal human case. Adhering to the seasonal influenza vaccine licensure regulations, we generated a H7N7/PR8 reassortant containing desired surface glycoprotein genes from the mallard viruses and internal genes of A/Puerto Rico/8/34 human vaccine strain (H1N1). Antigenic analysis revealed that the vaccine candidate virus confers broad antigenic cross-reactivity against contemporary Eurasian and the North American H7 subtype human isolates. Mice immunized with formalin inactivated (FI) H7N7/PR8 whole virus vaccine with or without aluminum hydroxide adjuvant conferred clinical protection from mortality and reduced pulmonary replication of the NL219 challenge virus. The FI H7N7/PR8 whole virus vaccine also afforded cross-protection in mice at the pulmonary level against antigenically distinct North American LP A/Canada/444/04 (H7N3) human isolate. The vaccine candidate virus satisfied the agricultural safety requirements for chickens, proved safe in mice, and has entered in phase-I human clinical trial in the United States.     

Analysis of the immunogenicity and bioactivities of a split influenza A/H7N9 vaccine mixed with MF59 adjuvant in BALB/c mice

The H7N9 influenza virus caused significant mortality and morbidity in humans during an outbreak in China in 2013. A recombinant H7N9 influenza seed with hemagglutinin (HA) and neuraminidase (NA) gene segments from A/Zhejiang/DTID-ZJU01/2013(H7N9) and six internal protein gene segments from A/Puerto Rico/8/34(H1N1; PR8) were generated using reverse genetics. We sought to determine the immunogenic, protective properties, and mechanisms of a split avian influenza A/H7N9 vaccine mixed with MF59 adjuvant in comparison to vaccines that included other adjuvant. BALB/c mice were vaccinated with two doses of different amounts and combinations of this novel A/ZJU01/PR8/2013 split vaccine with adjuvant. Mice were subsequently challenged with A/Zhejiang/DTID-ZJU01/2013(H7N9) by intranasal inoculation. We verified that MF59 enhanced the HI, MN, and IgG antibody titers to influenza antigens. Compared with alum, MF59 could more potentially induce humoral immune responses and Th2 cytokine production after virus infection, while both MF59 and alum can slightly increase NK cell activity. This split H7N9 influenza vaccine with MF59 adjuvant could effectively induce antibody production and protect mice from H7N9 virus challenge. We have selected this vaccine for manufacture and future clinical studies to protect humans from H7N9 virus infection.     

An Advax-CpG55.2 adjuvanted recombinant hemagglutinin vaccine provides immunity against H7N9 influenza in adult and neonatal mice

There is a major unmet need for strategies to improve the immunogenicity and effectiveness of pandemic influenza vaccines, particularly in poor responder populations such as neonates. Recombinant protein approaches to pandemic influenza offer advantages over more traditional inactivated virus approaches, as they are free of problems such as egg adaptation or need for high level biosecurity containment for manufacture. However, a weakness of recombinant proteins is their low immunogenicity. We asked whether the use of an inulin polysaccharide adjuvant (Advax) alone or combined with a TLR9 agonist (CpG55.2) would enhance the immunogenicity and protection of a recombinant hemagglutinin vaccine against H7N9 influenza (rH7HA), including in neonatal mice. Advax adjuvant induced predominantly IgG1 responses against H7HA, whereas Advax-CpG55.2 adjuvant also induced IgG2a, IgG2b and IgG3 responses, consistent with the TLR9 agonist component inducing a Th1 bias. Advax-CpG55.2 adjuvanted rH7HA induced high serum neutralizing antibody titers in adult mice. In newborns it similarly overcame immune hypo-responsiveness and enhanced serum anti-rH7HA IgG levels in 7-day-old BALB/C and C57BL/6 mice. Immunized adult mice were protected against a lethal H7N9 virus challenge. When formulated with Advax-CpG55.2 adjuvant, greater protection was seen with rH7HA than with inactivated H7 whole virus antigen. Advax-CpG55.2 adjuvanted rH7HA represents a promising influenza vaccine platform for further development.     

Recombinant H7 hemagglutinin expressed in glycoengineered Pichia pastoris forms nanoparticles that protect mice from challenge with H7N9 influenza virus

Cases of H7N9 human infection caused by an avian-origin H7N9 virus emerged in eastern China in 2013, leading to the urgent requirement of developing an effective vaccine to reduce its pandemic potential. In this report, the full-length recombinant H7 protein (rH7) of A/Hangzhou/1/2013 (H7N9) virus was expressed by a glycoengineered Pichia pastoris system. The rH7 protein underwent complex glycosylation modifications and polymerized to nanoparticles of 30–50 nm in diameter. Recombinant H7 (1.9 µg) elicited a > 1:40 hemagglutination inhibition titer, and 3.75 µg rH7 protected 100% of the mice in the mice challenge model with 10-fold 50% lethal dose of the A/Shanghai/2/2013 (H7N9) rat lung-adapted strain. In conclusion, rH7 produced by the glycoengineered P. pastoris can be used for vaccination against the H7N9 virus, and provides an effective platform for the rapid production of future influenza vaccines.     

Avian influenza A (H7N9) virus infection in humans: Epidemiology,evolution, and pathogenesis

New human influenza A virus strains regularly emerge causing seasonal epidemics and occasional pandemics. Lately, several zoonotic avian influenza A strains have been reported to directly infect humans. In early 2013, a novel avian influenza A virus (H7N9) strain was discovered in China to cause severe respiratory disease in humans. Since then, over 450 human cases of H7N9 infection have been discovered and 165 of them have died. Multiple epidemiological, phylogenetic, in vivo, and in vitro studies have been done to determine the origin and pathogenesis of novel H7N9 strain. This article reviews the literature related to the epidemiology, evolution, and pathogenesis of the H7N9 strain since its discovery in February 2013 till August 2014. The data available so far indicate that H7N9 was originated by a two-step reassortment process in birds and transmitted to humans through direct contact with live-bird markets. H7N9 is a low-pathogenic avian virus and contains several molecular signatures for adaptation in mammals. The severity of the respiratory disease caused by novel H7N9 virus in humans can be partly attributed to the age, sex, and underlying medical conditions of the patients. A universal influenza vaccine is not available, though several strain-specific H7N9 candidate vaccine viruses have been developed. Further, novel H7N9 virus is resistant to antiviral drug amantadine and some H7N9 isolates have acquired the resistance to neuraminidase-inhibitors. Therefore, constant surveillance and prompt control measures combined with novel research approaches to develop alternative and effective anti-influenza strategies are needed to overcome influenza A virus.     

One-shot vaccination with an insect cell-derived low-dose influenza A H7 virus-like particle preparation protects mice against H7N9 challenge

Human infections with a novel influenza A H7N9 subtype virus were reported in China recently. The virus caused severe disease with high mortality rates and it raised concerns over its pandemic potential. Here, we assessed in the mouse model protective efficacy of single immunisations with low vaccine doses of insect cell-derived H7 virus-like particles, consisting of hemagglutinin and matrix protein. Vaccinated mice were fully protected and survived a stringent lethal challenge (100 mLD50) with H7N9, even after a single, unadjuvanted, low vaccine dose (0.03 μg). Serum analysis revealed broad reactivity and hemagglutination inhibition activity across a panel of divergent H7 strains. Moreover, we detected significant levels of cross-reactivity to related group 2 hemagglutinins. These data demonstrate that virus-like particle vaccines have the potential to induce broadly protective immunity against the novel H7N9 virus and a variety of other H7 strains.     

Protective efficacy of an inactivated Eurasian avian-like H1N1 swine influenza vaccine against homologous H1N1 and heterologous H1N1 and H1N2 viruses in mice

Eurasian avian-like H1N1 (EA H1N1) swine influenza viruses are prevalent in pigs in Europe and Asia, but occasionally cause human infection, which raises concern about their pandemic potential. Here, we produced a whole-virus inactivated vaccine with an EA H1N1 strain (A/swine/Guangxi/18/2011, SW/GX/18/11) and evaluated its efficacy against homologous H1N1 and heterologous H1N1 and H1N2 influenza viruses in mice. A strong humoral immune response, which we measured by hemagglutination inhibition (HI) and virus neutralization (VN), was induced in the vaccine-inoculated mice upon challenge. The inactivated SW/GX/18/11 vaccine provided complete protection against challenge with homologous SW/GX/18/11 virus in mice and provided effective protection against challenge with heterologous H1N1 and H1N2 viruses with distinctive genomic combinations. Our findings suggest that this EA H1N1 vaccine can provide protection against both homologous H1N1 and heterologous H1N1 or H1N2 virus infection. As such, it is an excellent vaccine candidate to prevent H1N1 swine influenza.     

Protective efficacy of a polyvalent influenza A DNA vaccine against both homologous (H1N1pdm09) and heterologous (H5N1) challenge in the ferret model

This study describes the protective efficacy of a novel influenza plasmid DNA vaccine in the ferret challenge model. The rationally designed polyvalent influenza DNA vaccine encodes haemagglutinin and neuraminidase proteins derived from less glycosylated pandemic H1N1 (2009) and H3N2 (1968) virus strains as well as the nucleoprotein (NP) and matrix proteins (M1 and M2) from a different pandemic H1N1 (1918) strain. Needle-free intradermal immunisation with the influenza DNA vaccine protected ferrets against homologous challenge with an H1N1pdm09 virus strain, demonstrated by restriction of viral replication to the upper respiratory tract and reduced duration of viral shedding post-challenge. Breadth of protection was demonstrated in two heterologous efficacy experiments in which animals immunised with the influenza DNA vaccine were protected against challenge with a highly pathogenic avian influenza H5N1 virus strain with reproducible survival and clinical outcomes.     

Manipulation of neuraminidase packaging signals and hemagglutinin residues improves the growth of A/Anhui/1/2013 (H7N9) influenza vaccine virus yield in eggs

In 2013, a novel avian-origin H7N9 influenza A virus causing severe lower respiratory tract disease in humans emerged in China, with continued sporadic cases. An effective vaccine is needed for this virus in case it acquires transmissibility among humans; however, PR8-based A/Anhui/1/2013 (Anhui/1, H7N9), a WHO-recommended H7N9 candidate vaccine virus (CVV) for vaccine production, does not replicate well in chicken eggs, posing an obstacle to egg-based vaccine production. To address this issue, we explored the possibility that PR8’s hemagglutinin (HA) and neuraminidase (NA) packaging signals mediate improvement of Anhui/1 CVV yield in eggs. We constructed chimeric HA and NA genes having the coding region of Anhui/1 HA and NA flanked by the 5′ and 3′ packaging signals of PR8’s HA and NA, respectively. The growth of CVVs containing the chimeric HA was not affected, but that of those containing the chimeric NA gene grew in embryonated chicken eggs with a more than 2-fold higher titer than that of WT CVV. Upon 6 passages in eggs further yield increase was achieved although this was not associated with any changes in the chimeric NA gene. The HA of the passaged CVV, did, however, exhibit egg-adaptive mutations and one of them (HA-G218E) improved CVV growth in eggs without significantly changing antigenicity. The HA-G218E substitution and a chimeric NA, thus, combine to provide an Anhui/1 CVV with properties more favorable for vaccine manufacture.     

Evaluation of age-related differences in the immunogenicity of a G9 H9N2 influenza vaccine

Avian influenza A/H9N2 viruses can infect people and are viruses considered to be a potential pandemic threat. Prior studies with an inactivated G1 clade H9N2 vaccine reported that persons born before 1968 were more likely to have an immune response than younger subjects. We performed a randomized, double-blind trial to evaluate whether immune responses following immunization with an inactivated, unadjuvanted influenza G9 H9N2 vaccine prepared from A/chicken/Hong Kong/G9/97 virus were more frequent in persons born in 1964 or earlier (44-59 years) than in those born in 1970 or later (18-38 years). One hundred twenty one persons were randomized to receive two doses of either 7.5- or 30-mcg of hemagglutinin intramuscularly. Post-vaccination serum antibody responses as measured by hemagglutination inhibition and microneutralization were either similar in the two age cohorts or greater in the younger age group. Persons born before 1968 were not more likely to respond to a G9 H9N2 influenza vaccine than persons born in 1970 or later.     

Safety and immunogenicity of an 8 year interval heterologous prime-boost influenza A/H7N7-H7N9 vaccination

Background

Influenza A/H7N9 viruses are undergoing antigenic drift since their emergence in 2013, and vaccination strategies are needed for pandemic preparedness. Two doses of adjuvanted monovalent inactivated influenza A/H7N9 vaccine (IIV1 A/H7N9) are needed for optimal serological responses. However, administering 2 doses in a pandemic setting might be challenging. We evaluated the immunogenicity of “boosting” with IIV1 A/H7N9 in subjects “primed” 8?years previously with IIV1 A/H7N7.

Characterization of an influenza A H5N2 reassortant as a candidate for live-attenuated and inactivated vaccines against highly pathogenic H5N1 viruses with pandemic potential

We generated a high-growth 7:1 reassortant (Len17/H5) that contained the hemagglutinin (HA) gene from non-pathogenic A/Duck/Potsdam/1402-6/86 (H5N2) virus and other genes from the cold-adapted (ca) attenuated A/Leningrad/134/17/57 (H2H2) strain. Len17/H5 demonstrated an attenuated phenotype in mice and did not infect chickens. Mice administered Len17/H5 either as a live-attenuated intranasal vaccine or as an inactivated intramuscular vaccine were substantially protected from lethal challenge with highly pathogenic A/Hong Kong/483/97 (H5N1) virus and were protected from pulmonary infection with antigenically distinct A/Hong Kong/213/2003 (H5N1) virus. The cross-protective effect correlated with the levels of virus-specific mucosal IgA and/or serum IgG antibodies. Our results suggest a new strategy of using classical genetic reassortment between a high-growth ca H2N2 strain and antigenically related non-pathogenic avian viruses to prepare live-attenuated and inactivated vaccines for influenza pandemic.     

Transcutaneous immunization via dissolving microneedles protects mice from lethal influenza H7N9 virus challenge

Avian influenza H7N9 virus has first emerged in 2013 and since then has spread in China in five seasonal waves. In humans, influenza H7N9 virus infection is associated with a high fatality rate; thus, an effective vaccine for this virus is needed. In the present study, we evaluated the usefulness of dissolving microneedles (MNs) loaded with influenza H7N9 vaccine in terms of the dissolution time, insertion capacity, insertion depth, and structural integrity of H7N9 virus in vitro. Our in vitro results showed MNs dissolved within 6 mins. The depth of skin penetration was 270 µm. After coating with a matrix material solution, the H7N9 proteins were agglomerated. We detected the H7N9 delivery time and humoral immune response in vivo. In a mouse model, the antigen retention time was longer for MNs than for intramuscular (IM) injection. The humoral response showed that similar to IM administration, MN administration increased the levels of functional and systematic antibodies and protection against the live influenza A/Anhui/01/2013 virus (Ah01/H7N9). The protection level was determined by the analysis of pathological sections of infected lungs. MN and IM administration yielded results superior to those in the control group. Taken together, these findings demonstrate that the use of dissolving MNs to deliver influenza H7N9 vaccines is a promising immunization approach.