Mohammed Y.E. Chowdhury

Mucosal immunization with recombinant influenza hemagglutinin protein and poly gamma-glutamate/chitosan nanoparticles induces protection against highly pathogenic influenza A virus.

Intranasal administration of recombinant influenza hemagglutinin (rHA) antigen or inactivated virus with nanoparticles (NPs) composed of poly-γ-glutamic acid (γ-PGA) and chitosan which are safe, natural materials, and able to target the mucosal membrane as a mucosal adjuvant, could induce a high degree of protective mucosal immunity in the respiratory tract. Intranasal immunization with mixture of rHA antigen or inactivated virus and γ-PGA/chitosan nanoparticles (PC NPs) induced not only a high anti-HA immunoglobulin A (IgA) response in lung and IgG response in serum, including anti-HA neutralizing antibodies, but also an influenza virus-specific cell-mediated immune response. Also, PC NPs could function as a potential mucosal adjuvant when it was compared with the well-known mucosal adjuvant, cholera toxin (CT). Intranasal administration of rHA antigen or inactivated virus with PC NPs protected mice against challenge with a lethal dose of the highly pathogenic influenza A H5N1 virus. These results suggested that application of PC NPs with a subunit antigen of influenza produced by prokaryotic expression system provides several solutions to the current problems of the influenza vaccines using inactivated influenza virus. Moreover, our finding about a sufficient function of PC NPs to elevate vaccine efficacy led us to consider that it can be useful in clinical applications as a potent mucosal adjuvant with safety.

Mucosal vaccine delivery: Current state and a pediatric perspective

Most childhood infections occur via the mucosal surfaces, however, parenterally delivered vaccines are unable to induce protective immunity at these surfaces. In contrast, delivery of vaccines via the mucosal routes can allow antigens to interact with the mucosa-associated lymphoid tissue (MALT) to induce both mucosal and systemic immunity. The induced mucosal immunity can neutralize the pathogen on the mucosal surface before it can cause infection. In addition to reinforcing the defense at mucosal surfaces, mucosal vaccination is also expected to be needle-free, which can eliminate pain and the fear of vaccination. Thus, mucosal vaccination is highly appealing, especially for the pediatric population. However, vaccine delivery across mucosal surfaces is challenging because of the different barriers that naturally exist at the various mucosal surfaces to keep the pathogens out. There have been significant developments in delivery systems for mucosal vaccination. In this review we provide an introduction to the MALT, highlight barriers to vaccine delivery at different mucosal surfaces, discuss different approaches that have been investigated for vaccine delivery across mucosal surfaces, and conclude with an assessment of perspectives for mucosal vaccination in the context of the pediatric population.

Mucosal vaccination with recombinant Lactobacillus casei-displayed CTA1-conjugated consensus matrix protein-2 (sM2) induces broad protection against divergent influenza subtypes in BALB/c mice.

To develop a safe and effective mucosal vaccine against pathogenic influenza viruses, we constructed recombinant Lactobacillus casei strains that express conserved matrix protein 2 with (pgsA-CTA1-sM2/L. casei) or without (pgsA-sM2/L. casei) cholera toxin subunit A1 (CTA1) on the surface. The surface localization of the fusion protein was verified by cellular fractionation analyses, flow cytometry and immunofluorescence microscopy. Oral and nasal inoculations of recombinant L. casei into mice resulted in high levels of serum immunoglobulin G (IgG) and mucosal IgA. However, the conjugation of cholera toxin subunit A1 induced more potent mucosal, humoral and cell-mediated immune responses. In a challenge test with 10 MLD50 of A/EM/Korea/W149/06(H5N1), A/Puerto Rico/8/34(H1N1), A/Aquatic bird /Korea/W81/2005(H5N2), A/Aquatic bird/Korea/W44/2005(H7N3), and A/Chicken/Korea/116/2004(H9N2) viruses, the recombinant pgsA-CTA1-sM2/L. casei provided better protection against lethal challenges than pgsA-sM2/L. casei, pgsA/L. casei and PBS in mice. These results indicate that mucosal immunization with recombinant L. casei expressing CTA1-conjugated sM2 protein on its surface is an effective means of eliciting protective immune responses against diverse influenza subtypes.

Prokaryote-expressed M2e protein improves H9N2 influenza vaccine efficacy and protection against lethal influenza a virus in mice

BackgroundInfluenza vaccines are prepared annually based on global epidemiological surveillance data. However, since there is no method by which to predict the influenza strain that will cause the next pandemic, the demand to develop new vaccination strategies with broad cross-reactivity against influenza viruses are clearly important. The ectodomain of the influenza M2 protein (M2e) is an attractive target for developing a vaccine with broad cross-reactivity. For these reasons, we investigated the efficacy of an inactivated H9N2 virus vaccine (a-H9N2) mixed with M2e (1xM2e or 4xM2e) proteins expressed in Escherichia coli, which contains the consensus of sequence the extracellular domain of matrix 2 (M2e) of A/chicken/Vietnam/27262/09 (H5N1) avian influenza virus, and investigated its humoral immune response and cross-protection against influenza A viruses.ResultsMice were intramuscularly immunized with a-H9N2, 1xM2e alone, 4xM2e alone, a-H9N2/1xM2e, or a-H9N2/4xM2e. Three weeks post-vaccination, mice were challenged with lethal homologous (A/ chicken /Korea/ma163/04, H9N2) or heterosubtypic virus (A/Philippines/2/82, H3N2 and A/aquatic bird/Korea/maW81/05, H5N2). Our studies demonstrate that the survival of mice immunized with a-H9N2/1xM2e or with a-H9N2/4xM2e (100% survival) was significantly higher than that of mouse-adapted H9N2 virus-infected mice vaccinated with 1xM2e alone or with 4xM2e alone (0% survival). We also evaluated the protective efficacy of the M2e + vaccine against infection with mouse-adapted H5N2 influenza virus. Protection from death in the control group (0% survival) was similar to that of the 1×M2e alone and 4xM2e alone-vaccinated groups (0% survival). Only 40% of mice vaccinated with vaccine alone survived challenge with H5N2, while the a-H9N2/1×M2e and a-H9N2/4×M2e groups showed 80% and 100% survival following mouse-adapted H5N2 challenge, respectively. We also examined cross-protection against human H3N2 virus and found that the a-H9N2/1×M2e group displayed partial cross-protection against H3N2 (40% survival), whereas vaccine alone, 1×M2e alone, 4×M2e alone, or H9N2/1×M2e groups showed incomplete protection (0% survival) in response to challenge with a lethal dose of human H3N2 virus.ConclusionsTaken together, these results suggest that prokaryote-expressed M2e protein improved inactivated H9N2 virus vaccine efficacy and achieved cross-protection against lethal influenza A virus infection in mice.

The highly conserved HA2 protein of the influenza a virus induces a cross protective immune response

Existing influenza vaccines protect mostly homologous subtypes and acted most effectively only when well matched to the circulating strain. Immunization with an updated vaccine is therefore necessary to maintain long-term protection and the development of a broadly protective influenza vaccine against the threat of pandemic outbreak. The highly conserved HA2 glyco-polypeptide (HA2 gp) is a promising new candidate for such an influenza vaccine. Helical domain and the fusion peptide (residues 15-137) of surface antigen from influenza A subtype A/EM/Korea/W149/06 (H5N1) was used to assess the potentiality of HA2 vaccination against multiple subtypes of the influenza viruses. The construct, named H5HA2 was expressed in Escherichia coli and allowed to refold from inclusion bodies. Purified proteins were used to investigate the immunogenicity of H5HA2 and its potential for cross protection. The immunization of mice with H5HA2 induced HA2 antibodies, HA2 specific T-cell responses, and protection against homologous A/EM/Korea/W149/06 (H5N1) influenza. Immunized mice were also protected from two distinct heterosubtypes of influenza: A/Puerto Rico/1/34(H1N1) and bird/Korea/w81/2005(H5N2). Results suggest that recombinant proteins based on the highly conserved residues within HA2 are candidates for the development of vaccines against pandemic outbreaks of emergent influenza variants.

Mucosally administered Lactobacillus surface-displayed influenza antigens (sM2 and HA2) with cholera toxin subunit A1 (CTA1) Induce broadly protective immune responses against divergent influenza subtypes

The development of a universal influenza vaccine that provides broad cross protection against existing and unforeseen influenza viruses is a critical challenge. In this study, we constructed and expressed conserved sM2 and HA2 influenza antigens with cholera toxin subunit A1 (CTA1) on the surface of Lactobacillus casei (pgsA-CTA1sM2HA2/L. casei). Oral and nasal administrations of recombinant L. casei into mice resulted in high levels of serum immunoglobulin G (IgG) and their isotypes (IgG1 & IgG2a) as well as mucosal IgA. The mucosal administration of pgsA-CTA1sM2HA2/L. casei may also significantly increase the levels of sM2- or HA2-specific cell-mediated immunity because increased release of both IFN-γ and IL-4 was observed. The recombinant pgsA-CTA1sM2HA2/L. casei provided better protection of BALB/c mice against 10 times the 50% mouse lethal doses (MLD50) of homologous A/EM/Korea/W149/06(H5N1) or A/Aquatic bird/Korea/W81/2005 (H5N2) and heterologous A/Puerto Rico/8/34(H1N1), or A/Chicken/Korea/116/2004(H9N2) or A/Philippines/2/08(H3N2) viruses, compared with L. casei harboring sM2HA2 and also the protection was maintained up to seven months after administration. These results indicate that recombinant L. casei expressing the highly conserved sM2, HA2 of influenza and CTA1 as a mucosal adjuvant could be a potential mucosal vaccine candidate or tool to protect against divergent influenza viruses for human and animal.

Induction of type I interferon by high-molecular poly-γ-glutamate protects B6.A2G-Mx1 mice against influenza A virus

In addition to development of vaccines and synthetic antiviral drugs, recent studies have advocated the use of natural substances that inhibit or prevent viral infections. High-molecular-weight poly-γ-glutamate (HM-γ-PGA) produced by Bacillus subtilis chungkookjang was evaluated for anti-influenza virus activity. HM-γ-PGA induced type I interferons (IFNs), which in turn stimulated expression of Myxovirus resistant 1 protein and IFN-related proteins in vitro. In the B6.A2G-Mx1 mouse model, which mimics the innate immune system of humans, treatment with HM-γ-PGA enhanced the antiviral state of mice and protected them against highly pathogenic influenza A virus. Naturally synthesized HM-γ-PGA has potent anti-influenza activity and may be a useful means for control of influenza virus.

Heterosubtypic protective immunity against widely divergent influenza subtypes induced by fusion protein 4sM2 in BALB/c mice

BackgroundRegular reformulation of currently available vaccines is necessary due to the unpredictable variability of influenza viruses. Therefore, vaccine based on a highly conserved antigen with capability of induction of effective immune responses could be a potential solution. Influenza matrix protein-2 (M2) is highly conserved across influenza subtypes and a promising candidate for a broadly protective influenza vaccine. For the enhancement of broad protection, four tandem copies of consensus M2 gene containing extracellular (ED) and cytoplasmic (CD) without the trans-membrane domain (TM) reconstituted from H1N1, H5N1 and H9N2 influenza viruses were linked and named as 4sM2. The construct was effectively expressed in Escherichia coli, purified and proteins were used to immunize BALB/c mice. Humoral and cell-mediated immune responses were investigated following administration.ResultsMice were intramuscularly immunized with 4sM2 protein 2 times at 2 weeks interval. Two weeks after the last immunization, first humoral and cell mediated immune response specific to sM2 protein were evaluated and the mice were challenged with a lethal dose (10MLD50) of divergent subtypes A/EM/Korea/W149/06(H5N1), A/PR/8/34(H1N1), A/Aquatic bird/Korea/W81/2005(H5N2), A/Aquatic bird/Korea/W44/2005(H7N3), and A/Chicken/Korea/116/2004(H9N2) viruses. The efficacy of 4sM2 was evaluated by determining survival rates, body weights and residual lung viral titers. Our studies demonstrate that the survival of mice immunized with 4sM2 was significantly higher (80–100% survival) than that of unimmunized mice (0% survival). We also examined the long lasting protection against heterosubtype H5N2 virus and found that mice vaccinated with 4sM2 displayed 80% of protection even after 6 months of final vaccination.ConclusionTaken together, these results suggest that prokaryotic expressed multimeric sM2 protein achieved cross protection against lethal infection of divergent influenza subtypes which are lasting for the long time.

Antiviral Effects of Novel Herbal Medicine KIOM-C, on Diverse Viruses

In order to identify new potential antiviral agents, recent studies have advocated thorough testing of herbal medicines or natural substances that are traditionally used to prevent viral infections. Antiviral activities and the mechanism of action of the total aqueous extract preparation of KIOM-C, a novel herbal medicine, against diverse types of viruses were investigated. In vitro antiviral activity against A/Puerto Rico/8/34 (H1N1) (PR8), vesicular stomatitis virus (VSV), and Newcastle disease virus (NDV) through the induction of type-I interferon related protein phosphorylation and up-regulation of pro-inflammatory cytokines in murine macrophage cells (RAW264.7) were determined. In vivo, KIOM-C-treated BALB/c mice showed higher survivability and lower lung viral titers when challenged with A/Aquatic bird/Korea/W81/2005 (H5N2), A/PR/8/34(H1N1), A/Aquatic bird/Korea/W44/2005(H7N3) or A/Chicken/Korea/116 /2004(H9N2) influenza subtypes in contrast with the non-treated group. The present study revealed that total aqueous extract preparation of KIOM-C stimulates an antiviral state in murine macrophage cells and in mice leading to inhibition of viral infection and protection against lethal challenges.

Mucosal vaccination of conserved sM2, HA2 and cholera toxin subunit A1 (CTA1) fusion protein with poly gamma-glutamate/chitosan nanoparticles (PC NPs) induces protection against divergent influenza subtypes

To develop a safe and effective mucosal vaccine that broad cross protection against seasonal or emerging influenza A viruses, we generated a mucosal influenza vaccine system combining the highly conserved matrix protein-2 (sM2), fusion peptide of hemagglutinin (HA2), the well-known mucosal adjuvant cholera toxin subunit A1 (CTA1) and poly-γ-glutamic acid (γ-PGA)-chitosan nanoparticles (PC NPs), which are safe, natural materials that are able to target the mucosal membrane as a mucosal adjuvant. The mucosal administration of sM2HA2CTA1/PC NPs could induce a high degree of systemic immunity (IgG and IgA) at the site of inoculation as well as at remote locations and also significantly increase the levels of sM2- or HA2-specific cell-mediated immune response. In challenge tests in BALB/c mice with 10 MLD50 of A/EM/Korea/W149/06(H5N1), A/Puerto Rico/8/34(H1N1), A/Aquatic bird/Korea/W81/2005(H5N2), A/Aquatic bird/Korea/W44/2005 (H7N3) or A/Chicken/Korea/116/2004(H9N2) viruses, the recombinant sM2HA2CTA1/PC NPs provided cross protection against divergent lethal influenza subtypes and also the protection was maintained up to six months after vaccination. Thus, sM2HA2CTA1/PC NPs could be a promising strategy for a universal influenza vaccine.