Bivalent vaccination with NA1 and NA2 neuraminidase virus-like particles is protective against challenge with H1N1 and H3N2 influenza A viruses in a murine model.

Neuraminidase (NA) is the second most abundant glycoprotein on the surface of influenza A viruses (IAV). Neuraminidase type 1 (NA1) based virus-like particles (VLPs) have previously been shown to protect against challenge with H1N1 and H3N2 IAV. In this study, we produced neuraminidase type 2 (NA2) VLPs derived from the sequence of the seasonal IAV A/Perth/16/2009. Intramuscular vaccination of mice with NA2 VLPs induced high anti-NA serum IgG levels capable of inhibiting NA activity. NA2 VLP vaccination protected against mortality in a lethal A/Hong Kong/1/1968 (H3N2) virus challenge model, but not against lethal challenge with A/California/04/2009 (H1N1) virus. However, bivalent vaccination with NA1 and NA2 VLPs demonstrated no antigenic competition in anti-NA IgG responses and protected against lethal challenge with H1N1 and H3N2 viruses. Here we demonstrate that vaccination with NA VLPs is protective against influenza challenge and supports focusing on anti-NA responses in the development of future vaccination strategies.

Intranasal vaccination with influenza HA/GO-PEI nanoparticles provides immune protection against homo- and heterologous strains.

Intranasal (i.n.) immunization is a promising vaccination route for infectious respiratory diseases such as influenza. Recombinant protein vaccines can overcome the safety concerns and long production phase of virus-based influenza vaccines. However, soluble protein vaccines are poorly immunogenic if administered by an i.n. route. Here, we report that polyethyleneimine-functionalized graphene oxide nanoparticles (GP nanoparticles) showed high antigen-loading capacities and superior immunoenhancing properties. Via a facile electrostatic adsorption approach, influenza hemagglutinin (HA) was incorporated into GP nanoparticles and maintained structural integrity and antigenicity. The resulting GP nanoparticles enhanced antigen internalization and promoted inflammatory cytokine production and JAWS II dendritic cell maturation. Compared with soluble HA, GP nanoparticle formulations induced significantly enhanced and cross-reactive immune responses at both systemic sites and mucosal surfaces in mice after i.n. immunization. In the absence of any additional adjuvant, the GP nanoparticle significantly boosted antigen-specific humoral and cellular immune responses, comparable to the acknowledged potent mucosal immunomodulator CpG. The robust immune responses conferred immune protection against challenges by homologous and heterologous viruses. Additionally, the solid self-adjuvant effect of GP nanoparticles may mask the role of CpG when coincorporated. In the absence of currently approved mucosal adjuvants, GP nanoparticles can be developed into potent i.n. influenza vaccines, providing broad protection. With versatility and flexibility, the GP nanoplatform can be easily adapted for constructing mucosal vaccines for different respiratory pathogens.

Fabrication of microneedle patches with lyophilized influenza vaccine suspended in organic solvent.

Skin vaccination by microneedle (MN) patch simplifies the immunization process to increase access to vaccines for global health. Lyophilization has been widely used to stabilize vaccines and other biologics during storage, but is generally not compatible with the MN patch manufacturing processes. In this study, our goal was to develop a method to incorporate lyophilized inactivated H1N1 influenza vaccine into MN patches during manufacturing by suspending freeze-dried vaccine in anhydrous organic solvent during the casting process. Using a casting formulation containing chloroform and polyvinylpyrrolidone, lyophilized influenza vaccine maintained activity during manufacturing and subsequent storage for 3 months at 40 °C. Influenza vaccination using these MN patches generated strong immune responses in a murine model. This manufacturing process may enable vaccines and other biologics to be stabilized by lyophilization and administered via a MN patch.

Cutaneous vaccination ameliorates Zika virus-induced neuro-ocular pathology via reduction of anti-ganglioside antibodies.

Zika virus (ZIKV) causes moderate to severe neuro-ocular sequelae, with symptoms ranging from conjunctivitis to Guillain-Barré Syndrome (GBS). Despite the international threat ZIKV poses, no licensed vaccine exists. As ZIKV and DENV are closely related, antibodies against one virus have demonstrated the ability to enhance the other. To examine if vaccination can confer robust, long-term protection against ZIKV, preventing neuro-ocular pathology and long-term inflammation in immune-privileged compartments, BALB/c mice received two doses of unadjuvanted inactivated whole ZIKV vaccine (ZVIP) intramuscularly (IM) or cutaneously with dissolving microneedle patches (MNP). MNP immunization induced significantly higher B and T cell responses compared to IM vaccination, resulting in increased antibody titers with greater avidity for ZPIV as well as increased numbers of IFN-γ, TNF-α, IL- and IL-4 secreting T cells. When compared to IM vaccination, antibodies generated by cutaneous vaccination demonstrated greater neutralization activity, increased cross-reactivity with Asian and African lineage ZIKV strains (PRVABC59, FLR, and MR766) and Dengue virus (DENV) serotypes, limited ADE, and lower reactivity to GBS-associated gangliosides. MNP vaccination effectively controlled viremia and inflammation, preventing neuro-ocular pathology. Conversely, IM vaccination exacerbated ocular pathology, resulting in uncontrolled, long-term inflammation. Importantly, neuro-ocular pathology correlated with anti-ganglioside antibodies implicated in demyelination and GBS. This study highlights the importance of longevity studies in ZIKV immunization, and the need of exploring alternative vaccination platforms to improve the quality of vaccine-induced immune responses.

Intradermal Immunization of EBOV VLPs in Guinea Pigs Induces Broader Antibody Responses Against GP Than Intramuscular Injection.

Ebolavirus (EBOV) infection in humans causes severe hemorrhagic fevers with high mortality rates that range from 30 to 80% as shown in different outbreaks. Thus the development of safe and efficacious EBOV vaccines remains an important goal for biomedical research. We have shown in early studies that immunization with insect cell-produced EBOV virus-like particles (VLPs) is able to induce protect vaccinated mice against lethal EBOV challenge. In the present study, we investigated immune responses induced by Ebola VLPs via two different routes, intramuscular and intradermal immunizations, in guinea pigs. Analyses of antibody responses revealed that similar levels of total IgG antibodies against the EBOV glycoprotein (GP) were induced by the two different immunization methods. However, further characterization showed that the EBOV GP-specific antibodies induced by intramuscular immunization were mainly of the IgG2 subtype whereas both IgG1 and IgG2 antibodies against EBOV GP were induced by intradermal immunization. In contrast, antibody responses against the EBOV matrix protein VP40 induced by intramuscular or intradermal immunizations exhibited similar IgG1 and IgG2 profiles. More interestingly, we found that the sites that the IgG1 antibodies induced by intradermal immunizations bind to in GP are different from those that bind to the IgG2 antibodies induced by intramuscular immunization. Further analyses revealed that sera from all vaccinated guinea pigs exhibited neutralizing activity against Ebola GP-mediated HIV pseudovirion infection at high levels. Moreover, all EBOV VLP-vaccinated guinea pigs survived the challenge by a high dose (1000 pfu) of guinea pig-adapted EBOV, while all control guinea pigs immunized with irrelevant VLPs succumbed to the challenge. The induction of both IgG1 and IgG2 antibody responses that recognized broader sites in GP by intradermal immunization of EBOV VLPs indicates that this approach may represent a more advantageous route of vaccination against virus infection.

cGAMP/Saponin Adjuvant Combination Improves Protective Response to Influenza Vaccination by Microneedle Patch in an Aged Mouse Model.

Current strategies for improving protective response to influenza vaccines during immunosenescence do not adequately protect individuals over 65 years of age. Here, we used an aged mouse model to investigate the potential of co-delivery of influenza vaccine with the recently identified combination of a saponin adjuvant Quil-A and an activator of the STING pathway, 2'3 cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) via dissolving microneedle patches (MNPs) applied to skin. We demonstrate that synergy between the two adjuvant components is observed after their incorporation with H1N1 vaccine into MNPs as revealed by analysis of the immune responses in adult mice. Aged 21-month-old mice were found to be completely protected against live influenza challenge after vaccination with the MNPs adjuvanted with the Quil-A/cGAMP combination (5 µg each) and demonstrated significantly reduced morbidity compared to the observed responses in these mice vaccinated with unadjuvanted MNPs. Analysis of the lung lysates of the surviving aged mice post challenge revealed the lowest level of residual inflammation in the adjuvanted groups. We conclude that combining influenza vaccine with a STING pathway activator and saponin-based adjuvant in MNPs is a novel option for skin vaccination of the immunosenescent population, which is at high risk for influenza.

Extended delivery of vaccines to the skin improves immune responses.

Vaccines prevent 2-3 million childhood deaths annually; however, low vaccine efficacy and the resulting need for booster doses create gaps in immunization coverage. In this translational study, we explore the benefits of extended release of licensed vaccine antigens into skin to increase immune responses after a single dose in order to design improved vaccine delivery systems. By administering daily intradermal injections of inactivated polio vaccine according to six different delivery profiles, zeroth-order release over 28 days resulted in neutralizing antibody titers equivalent to two bolus vaccinations administered one month apart. Vaccinations following this profile also improved immune responses to tetanus toxoid and subunit influenza vaccine but not a live-attenuated viral vaccine, measles vaccine. Finally, using subunit influenza vaccine, we demonstrated that daily vaccination by microneedle patch induced a potent, balanced humoral immunity with an increased memory response compared to bolus vaccination. We conclude that extended presentation of antigen in skin via intradermal injection or microneedle patch can enhance immune responses and reduce the number of vaccine doses, thereby enabling increased vaccination efficacy.

Co-Delivery of M2e Virus-Like Particles with Influenza Split Vaccine to the Skin Using Microneedles Enhances the Efficacy of Cross Protection.

It is a high priority to develop a simple and effective delivery method for a cross-protective influenza vaccine. We investigated skin immunization by microneedle (MN) patch with human influenza split vaccine and virus-like particles containing heterologous M2 extracellular (M2e) domains (M2e5x virus-like particles (VLP)) as a cross-protective influenza vaccine candidate. Co-delivery of influenza split vaccine and M2e5x VLP to the skin by MN patch was found to confer effective protection against heterosubtypic influenza virus by preventing weight loss and reducing lung viral loads. Compared to intramuscular immunization, MN-based delivery of combined split vaccine and M2e5x VLPs shaped cellular immune responses toward T helper type 1 responses increasing IgG2a isotype antibodies as well as IFN-γ producing cells in mucosal and systemic sites. This study provides evidence that potential immunological and logistic benefits of M2e5x VLP with human influenza split vaccine delivered by MN patch can be used to develop an easy-to-administer cross-protective influenza vaccine.

Combination of STING Pathway Agonist With Saponin Is an Effective Adjuvant in Immunosenescent Mice.

There is an urgent need to improve protective responses to influenza vaccination in the elderly population, which is at especially high risk for adverse outcomes from influenza infection. Currently available inactivated vaccines provide limited protection, even when a 4-fold higher dose of the vaccine is administered. Adjuvants are often added to vaccines to boost protective efficacy. Here we describe a novel combination of an activator of the STING pathway, 2',3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) with a saponin adjuvant, that we found to be highly effective in boosting protective immunity from vaccination in an aged mouse model. Using this combination with a subunit influenza vaccine, we observed that survival of vaccinated 20 month-old mice after lethal challenge increased from 0 to 20% with unadjuvanted vaccine to 80-100%, depending on the vaccination route. Compared to unadjuvanted vaccine, the levels of vaccine-specific IgG and IgG2a increased by almost two orders of magnitude as early as 2 weeks after a single immunization with the adjuvanted formulation. By analyzing phosphorylation of interferon regulatory factor 3 (IRF3) in cell culture, we provide evidence that the saponin component increases access of exogenous cGAMP to the intracellular STING pathway. Our findings suggest that combining a STING activator with a saponin-based adjuvant increases the effectiveness of influenza vaccine in aged hosts, without having to increase dose or perform additional vaccinations. This study reports a novel adjuvant combination that (a) is more effective than current methods of boosting vaccine efficacy, (b) can be used to enhance efficacy of licensed influenza vaccines, and (c) results in effective protection using a single vaccine dose.

Heterosubtypic influenza protection elicited by double-layered polypeptide nanoparticles in mice.

Influenza is a persistent threat to public health. Here we report that double-layered peptide nanoparticles induced robust specific immunity and protected mice against heterosubtypic influenza A virus challenges. We fabricated the nanoparticles by desolvating a composite peptide of tandem copies of nucleoprotein epitopes into nanoparticles as cores and cross-linking another composite peptide of four tandem copies of influenza matrix protein 2 ectodomain epitopes to the core surfaces as a coating. Delivering the nanoparticles via dissolvable microneedle patch-based skin vaccination further enhanced the induced immunity. These peptide-only, layered nanoparticles demonstrated a strong antigen depot effect and migrated into spleens and draining (inguinal) lymph nodes for an extended period compared with soluble antigens. This increased antigen-presentation time correlated with the stronger immune responses in the nanoparticle-immunized group. The protection conferred by nanoparticle immunization was transferable by passive immune serum transfusion and depended partially on a functional IgG receptor FcγRIV. Using a conditional cell depletion, we found that CD8+ T cells were involved in the protection. The immunological potency and stability of the layered peptide nanoparticles indicate applications for other peptide-based vaccines and peptide drug delivery.

Intradermal immunization by Ebola virus GP subunit vaccines using microneedle patches protects mice against lethal EBOV challenge.

Development of a safe and efficacious filovirus vaccine is of high importance to public health. In this study, we compared immune responses induced by Ebola virus (EBOV) glycoprotein (GP) subunit vaccines via intradermal immunization with microneedle (MN) patches and the conventional intramuscular (IM) injection in mice, which showed that MN delivery of GP induced higher levels and longer lasting antibody responses against GP than IM injection. Further, we found that EBOV GP in formulation with a saponin-based adjuvant, Matrix-M, can be efficiently loaded onto MN patches. Co-delivery of Matrix-M with GP significantly enhanced induction of antibody responses by MN delivery, as also observed for IM injection. Results from challenge studies showed that all mice that received the GP/adjuvant formulation by MN or IM immunizations were protected from lethal EBOV challenge. Further, 4 out of 5 mice vaccinated by MN delivery of unadjuvanted GP also survived the challenge, whereas only 1 out of 5 mice vaccinated by IM injection of unadjuvanted GP survived the challenge. These results demonstrate that MN patch delivery of EBOV GP subunit vaccines, which is expected to enable improved safety and thermal stability, can confer effective protection against EBOV infection that is superior to IM vaccination.

Intradermal Vaccination With Adjuvanted Ebola Virus Soluble Glycoprotein Subunit Vaccine by Microneedle Patches Protects Mice Against Lethal Ebola Virus Challenge.

In this study, we investigated immune responses induced by purified Ebola virus (EBOV) soluble glycoprotein (sGP) subunit vaccines via intradermal immunization with microneedle (MN) patches in comparison with intramuscular (IM) injection in mice. Our results showed that MN delivery of EBOV sGP was superior to IM injection in eliciting higher levels and longer lasting antibody responses against EBOV sGP and GP antigens. Moreover, sGP-specific immune responses induced by MN or IM immunizations were effectively augmented by formulating sGP with a saponin-based adjuvant, and they were shown to confer complete protection of mice against lethal mouse-adapted EBOV (MA-EBOV) challenge. In comparison, mice that received sGP without adjuvant by MN or IM immunizations succumbed to lethal MA-EBOV challenge. These results show that immunization with EBOV sGP subunit vaccines with adjuvant by MN patches, which have been shown to provide improved safety and thermal stability, is a promising approach to protect against EBOV infection.

NAction! How Can Neuraminidase-Based Immunity Contribute to Better Influenza Virus Vaccines?

Neuraminidase is one of the two surface glycoproteins of influenza A and B viruses. It has enzymatic activity that cleaves terminal sialic acid from glycans, and that activity is essential at several points in the virus life cycle. While neuraminidase is a major target for influenza antivirals, it is largely ignored in vaccine development. Current inactivated influenza virus vaccines might contain neuraminidase, but the antigen quantity and quality are varied and not standardized. While there are data that show a protective role of anti-neuraminidase immunity, many questions remain unanswered. These questions, among others, concern the targeted epitopes or antigenic sites, the potential for antigenic drift, and, connected to that, the breadth of protection, differences in induction of immune responses by vaccination versus infection, mechanisms of protection, the role of mucosal antineuraminidase antibodies, stability, and the immunogenicity of neuraminidase in vaccine formulations. Reagents for analysis of neuraminidase-based immunity are scarce, and assays are not widely used for clinical studies evaluating vaccines. However, efforts to better understand neuraminidase-based immunity have been made recently. A neuraminidase focus group, NAction!, was formed at a Centers of Excellence for Influenza Research and Surveillance meeting at the National Institutes of Health in Bethesda, MD, to promote research that helps to understand neuraminidase-based immunity and how it can contribute to the design of better and broadly protective influenza virus vaccines. Here, we review open questions and knowledge gaps that have been identified by this group and discuss how the gaps can be addressed, with the ultimate goal of designing better influenza virus vaccines.

Stable incorporation of GM-CSF into dissolvable microneedle patch improves skin vaccination against influenza.

The widely used influenza subunit vaccine would benefit from increased protection rates in vulnerable populations. Skin immunization by microneedle (MN) patch can increase vaccine immunogenicity, as well as increase vaccination coverage due to simplified administration. To further increase immunogenicity, we used granulocyte-macrophage colony stimulating factor (GM-CSF), an immunomodulatory cytokine already approved for skin cancer therapy and cancer support treatment. GM-CSF has been shown to be upregulated in skin following MN insertion. The GM-CSF-adjuvanted vaccine induced robust and long-lived antibody responses cross-reactive to homosubtypic and heterosubtypic influenza viruses. Addition of GM-CSF resulted in increased memory B cell persistence relative to groups given influenza vaccine alone and led to rapid lung viral clearance following lethal infection with homologous virus in the mouse model. Here we demonstrate that successful incorporation of the thermolabile cytokine GM-CSF into MN resulted in improved vaccine-induced protective immunity holding promise as a novel approach to improved influenza vaccination. To our knowledge, this is the first successful incorporation of a cytokine adjuvant into dissolvable MNs, thus advancing and diversifying the rapidly developing field of MN vaccination technology.

Double-layered protein nanoparticles induce broad protection against divergent influenza A viruses.

Current influenza vaccines provide limited protection against circulating influenza A viruses. A universal influenza vaccine will eliminate the intrinsic limitations of the seasonal flu vaccines. Here we report methodology to generate double-layered protein nanoparticles as a universal influenza vaccine. Layered nanoparticles are fabricated by desolvating tetrameric M2e into protein nanoparticle cores and coating these cores by crosslinking headless HAs. Representative headless HAs of two HA phylogenetic groups are constructed and purified. Vaccinations with the resulting protein nanoparticles in mice induces robust long-lasting immunity, fully protecting the mice against challenges by divergent influenza A viruses of the same group or both groups. The results demonstrate the importance of incorporating both structure-stabilized HA stalk domains and M2e into a universal influenza vaccine to improve its protective potency and breadth. These potent disassemblable protein nanoparticles indicate a wide application in protein drug delivery and controlled release.

Vaccination with Combination DNA and Virus-Like Particles Enhances Humoral and Cellular Immune Responses upon Boost with Recombinant Modified Vaccinia Virus Ankara Expressing Human Immunodeficiency Virus Envelope Proteins.

Heterologous prime boost with DNA and recombinant modified vaccinia virus Ankara (rMVA) vaccines is considered as a promising vaccination approach against human immunodeficiency virus (HIV-1). To further enhance the efficacy of DNA-rMVA vaccination, we investigated humoral and cellular immune responses in mice after three sequential immunizations with DNA, a combination of DNA and virus-like particles (VLP), and rMVA expressing HIV-1 89.6 gp120 envelope proteins (Env). DNA prime and boost with a combination of VLP and DNA vaccines followed by an rMVA boost induced over a 100-fold increase in Env-specific IgG antibody titers compared to three sequential immunizations with DNA and rMVA. Cellular immune responses were induced by VLP-DNA and rMVA vaccinations at high levels in CD8 T cells, CD4 T cells, and peripheral blood mononuclear cells secreting interferon (IFN)-γ, and spleen cells producing interleukin (IL)-2, 4, 5 cytokines. This study suggests that a DNA and VLP combination vaccine with MVA is a promising strategy in enhancing the efficacy of DNA-rMVA vaccination against HIV-1.

Skin immunization by microneedle patch overcomes statin-induced suppression of immune responses to influenza vaccine.

Recent studies indicated that in elderly individuals, statin therapy is associated with a reduced response to influenza vaccination. The present study was designed to determine effects on the immune response to influenza vaccination induced by statin administration in a mouse model, and investigate potential approaches to improve the outcome of vaccination on the background of statin therapy. We fed middle aged BALB/c mice a high fat "western" diet (WD) alone or supplemented with atorvastatin (AT) for 14 weeks, and control mice were fed with the regular rodent diet. Mice were immunized with a single dose of subunit A/Brisbane/59/07 (H1N1) vaccine, either systemically or with dissolving microneedle patches (MNPs). We observed that a greater age-dependent decline in the hemagglutinin inhibition titers occurred in systemically-immunized mice than in MNP- immunized mice. AT dampened the antibody response in the animals vaccinated by either route of vaccine delivery. However, the MNP-vaccinated AT-treated animals had ~20 times higher total antibody levels to the influenza vaccine than the systemically vaccinated group one month postvaccination. We propose that microneedle vaccination against influenza provides an approach to ameliorate the immunosuppressive effect of statin therapy observed with systemic immunization.

H1N1 influenza virus infection results in adverse pregnancy outcomes by disrupting tissue-specific hormonal regulation.

Increased susceptibility to influenza virus infection during pregnancy has been attributed to immunological changes occurring before and during gestation in order to "tolerate" the developing fetus. These systemic changes are most often characterized by a suppression of cell-mediated immunity and elevation of humoral immune responses referred to as the Th1-Th2 shift. However, the underlying mechanisms which increase pregnant mothers' risk following influenza virus infection have not been fully elucidated. We used pregnant BALB/c mice during mid- to late gestation to determine the impact of a sub-lethal infection with A/Brisbane/59/07 H1N1 seasonal influenza virus on completion of gestation. Maternal and fetal health status was closely monitored and compared to infected non-pregnant mice. Severity of infection during pregnancy was correlated with premature rupture of amniotic membranes (PROM), fetal survival and body weight at birth, lung viral load and degree of systemic and tissue inflammation mediated by innate and adaptive immune responses. Here we report that influenza virus infection resulted in dysregulation of inflammatory responses that led to pre-term labor, impairment of fetal growth, increased fetal mortality and maternal morbidity. We observed significant compartment-specific immune responses correlated with changes in hormonal synthesis and regulation. Dysregulation of progesterone, COX-2, PGE2 and PGF2α expression in infected pregnant mice was accompanied by significant remodeling of placental architecture and upregulation of MMP-9 early after infection. Collectively these findings demonstrate the potential of a seasonal influenza virus to initiate a powerful pro-abortive mechanism with adverse outcomes in fetal health.