Marina De Filette

Universal Vaccine Based on Ectodomain of Matrix Protein 2 of Influenza A: Fc Receptors and Alveolar Macrophages Mediate Protection

The ectodomain of matrix protein 2 (M2e) of influenza A virus is an attractive target for a universal influenza A vaccine: the M2e sequence is highly conserved across influenza virus subtypes, and induced humoral anti-M2e immunity protects against a lethal influenza virus challenge in animal models. Clinical phase I studies with M2e vaccine candidates have been completed. However, the in vivo mechanism of immune protection induced by M2e-carrier vaccination is unclear. Using passive immunization experiments in wild-type, FcRγ−/−, FcγRI−/−, FcγRIII−/−, and (FcγRI, FcγRIII)−/− mice, we report in this study that Fc receptors are essential for anti-M2e IgG-mediated immune protection. M2e-specific IgG1 isotype Abs are shown to require functional FcγRIII for in vivo immune protection but other anti-M2e IgG isotypes can rescue FcγRIII−/− mice from a lethal challenge. Using a conditional cell depletion protocol, we also demonstrate that alveolar macrophages (AM) play a crucial role in humoral M2e-specific immune protection. Additionally, we show that adoptive transfer of wild-type AM into (FcγRI, FcγRIII)−/− mice restores protection by passively transferred anti-M2e IgG. We conclude that AM and Fc receptor-dependent elimination of influenza A virus-infected cells are essential for protection by anti-M2e IgG.

Universal M2 ectodomain-based influenza A vaccines: preclinical and clinical developments

Influenza vaccines used today are strain specific and need to be adapted every year to try and match the antigenicity of the virus strains that are predicted to cause the next epidemic. The strain specificity of the next pandemic is unpredictable. An attractive alternative approach would be to use a vaccine that matches multiple influenza virus strains, including multiple subtypes. In this review, we focus on the development and clinical potential of a vaccine that is based on the conserved ectodomain of matrix protein 2 (M2) of influenza A virus. Since 1999, a number of studies have demonstrated protection against influenza A virus challenge in animal models using chemical or genetic M2 external domain (M2e) fusion constructs. More recently, Phase I clinical studies have been conducted with M2e vaccine candidates, demonstrating their safety and immunogenicity in humans. Ultimately, and possibly in the near future, efficacy studies in humans should provide proof that this novel vaccine concept can mitigate epidemic and even pandemic influenza A virus infections.

An Influenza A Vaccine Based on Tetrameric Ectodomain of Matrix Protein 2

Matrix protein 2 (M2) of influenza A is a tetrameric type III membrane protein that functions as a proton-selective channel. The extracellular domain (M2e) has remained nearly invariable since the first human influenza strain was isolated in 1933. By linking a modified form of the leucine zipper of the yeast transcription factor GCN4 to M2e, we obtained a recombinant tetrameric protein, M2e-tGCN4. This protein mimics the quaternary structure of the ectodomain of the natural M2 protein. M2e-tGCN4 was purified, biochemically characterized, and used to immunize BALB/c mice. High M2e-specific serum IgG antibody titers were obtained following either intraperitoneal or intranasal administration. Immunized mice were protected fully against a potentially lethal influenza A virus challenge. Antibodies raised by M2e-tGCN4 immunization specifically bound to the surface of influenza-infected cells and to an M2-expressing cell line. Using a M2e peptide competition enzyme-linked immunosorbent assay with M2-expressing cells as target, we obtained evidence that M2e-tGCN4 induces antibodies that are specific for the native tetrameric M2 ectodomain. Therefore, fusion of an oligomerization domain to the extracellular part of a transmembrane protein allows it to mimic the natural quaternary structure and can promote the induction of oligomer-specific antibodies.

Recent progress in West Nile virus diagnosis and vaccination

West Nile virus (WNV) is a positive-stranded RNA virus belonging to the Flaviviridae family, a large family with 3 main genera (flavivirus, hepacivirus and pestivirus). Among these viruses, there are several globally relevant human pathogens including the mosquito-borne dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV) and West Nile virus (WNV), as well as tick-borne viruses such as tick-borne encephalitis virus (TBEV). Since the mid-1990s, outbreaks of WN fever and encephalitis have occurred throughout the world and WNV is now endemic in Africa, Asia, Australia, the Middle East, Europe and the Unites States. This review describes the molecular virology, epidemiology, pathogenesis, and highlights recent progress regarding diagnosis and vaccination against WNV infections.

Polymeric multilayer capsule-mediated vaccination induces protective immunity against cancer and viral infection

Recombinant antigens hold high potential to develop vaccines against lethal intracellular pathogens and cancer. However, they are poorly immunogenic and fail to induce potent cellular immunity. In this paper, we demonstrate that polymeric multilayer capsules (PMLC) strongly increase antigen delivery toward professional antigen-presenting cells in vivo, including dendritic cells (DCs), macrophages, and B cells, thereby enforcing antigen presentation and stimulating T cell proliferation. A thorough analysis of the T cell response demonstrated their capacity to induce IFN-γ secreting CD4 and CD8 T cells, in addition to follicular T-helper cells, a recently identified CD4 T cell subset supporting antibody responses. On the B cell level, PMLC-mediated antigen delivery promoted the formation of germinal centers, resulting in increased numbers of antibody-secreting plasma cells and elevated antibody titers. The functional relevance of the induced immune responses was validated in murine models of influenza and melanoma. On a mechanistic level, we have demonstrated the capacity of PMLC to activate the NALP3 inflammasome and trigger the release of the potent pro-inflammatory cytokine IL-1β. Finally, using DC-depleted mice, we have identified DCs as the key mediators of the immunogenic properties of PMLC.

Universal influenza A M2e-HBc vaccine protects against disease even in the presence of pre-existing anti-HBc antibodies

The extracellular domain of influenza A virus matrix protein 2 (M2e) is strongly conserved. Therefore, vaccines based on M2e can induce broad-spectrum immunity against influenza. We have mainly used recombinant virus-like particles derived from Hepatitis B virus core (HBc) as carrier for efficacious presentation of the M2e antigen. Here, we address whether pre-existing HBc-specific immunity interferes with the protective immune response obtained by M2e-HBc vaccination. Anti-HBc antibodies were induced by immunizing mice with unsubstituted HBc virus-like particles in the presence of two different adjuvants. We demonstrate that pre-existing HBc-specific antibodies affect neither the induction of M2e-specific antibody responses to vaccination with M2e-HBc particles, nor the protective efficacy of the resulting response. These results suggest that vaccination with M2e-HBc can induce protective anti-M2e antibodies even in anti-HBc positive individuals. The implications of these findings are discussed in the context of the clinical development of an M2e-based universal influenza vaccine, which recently successfully completed a Phase I trial.

Nanobodies With In Vitro Neutralizing Activity Protect Mice Against H5N1 Influenza Virus Infection

Influenza A virus infections impose a recurrent and global disease burden. Current antivirals against influenza are not always effective. We assessed the protective potential of monovalent and bivalent Nanobodies (Ablynx) against challenge with this virus. These Nanobodies were derived from llamas and target H5N1 hemagglutinin. Intranasal administration of Nanobodies effectively controlled homologous influenza A virus replication. Administration of Nanobodies before challenge strongly reduced H5N1 virus replication in the lungs and protected mice from morbidity and mortality after a lethal challenge with H5N1 virus. The bivalent Nanobody was at least 60-fold more effective than the monovalent Nanobody in controlling virus replication. In addition, Nanobody therapy after challenge strongly reduced viral replication and significantly delayed time to death. Epitope mapping revealed that the VHH Nanobody binds to antigenic site B in H5 hemagglutinin. Because Nanobodies are small, stable, and simple to produce, they are a promising, novel therapeutic agent against influenza.

M2e-displaying virus-like particles with associated RNA promote T helper 1 type adaptive immunity against influenza A.

The ectodomain of influenza A matrix protein 2 (M2e) is a candidate for a universal influenza A vaccine. We used recombinant Hepatitis B core antigen to produce virus-like particles presenting M2e (M2e-VLPs). We produced the VLPs with and without entrapped nucleic acids and compared their immunogenicity and protective efficacy. Immunization of BALB/c mice with M2e-VLPs containing nucleic acids induced a stronger, Th1-biased antibody response compared to particles lacking nucleic acids. The former also induced a stronger M2e-specific CD4+ T cell response, as determined by ELISPOT. Mice vaccinated with alum-adjuvanted M2e-VLPs containing the nucleic acid-binding domain were better protected against influenza A virus challenge than mice vaccinated with similar particles lacking this domain, as deduced from the loss in body weight following challenge with X47 (H3N2) or PR/8 virus. Challenge of mice that had been immunized with M2e-VLPs with or without nucleic acids displayed significantly lower mortality, morbidity and lung virus titers than control-immunized groups. We conclude that nucleic acids present in M2e-VLPs correlate with improved immune protection.

Innate immune response and programmed cell death following carrier-mediated delivery of unmodified mRNA to respiratory cells

In this report we show that carrier-mediated delivery of mRNA may activate TLR3 signaling in respiratory cells. This activation of the innate immune system was accompanied with a massive production of type 1 interferons and other immunostimulating cytokines. The recognition of mRNA by the innate immune system was also associated with cell death, which proceeded in human respiratory cells via pyroptosis, a form of programmed cell death mediated by substantial overexpression of caspase-1. This indicated that the delivered mRNA is most likely also recognized by NOD-like receptors which regulate caspase-1 production. The viability of murine respiratory cells was less affected by mRNA transfection, which is in line with the lower transfection efficiency, lower innate immune response and the absence of a massive caspase-1 upregulation in these cells. Finally, we also demonstrated that the recognition of the delivered mRNA by the innate immune system had a negative effect on mRNA translation.

Vaccination of mice using the West Nile virus E-protein in a DNA prime-protein boost strategy stimulates cell-mediated immunity and protects mice against a lethal challenge.

West Nile virus (WNV) is a mosquito-borne flavivirus that is endemic in Africa, the Middle East, Europe and the United States. There is currently no antiviral treatment or human vaccine available to treat or prevent WNV infection. DNA plasmid-based vaccines represent a new approach for controlling infectious diseases. In rodents, DNA vaccines have been shown to induce B cell and cytotoxic T cell responses and protect against a wide range of infections. In this study, we formulated a plasmid DNA vector expressing the ectodomain of the E-protein of WNV into nanoparticles by using linear polyethyleneimine (lPEI) covalently bound to mannose and examined the potential of this vaccine to protect against lethal WNV infection in mice. Mice were immunized twice (prime – boost regime) with the WNV DNA vaccine formulated with lPEI-mannose using different administration routes (intramuscular, intradermal and topical). In parallel a heterologous boost with purified recombinant WNV envelope (E) protein was evaluated. While no significant E-protein specific humoral response was generated after DNA immunization, protein boosting of DNA-primed mice resulted in a marked increase in total neutralizing antibody titer. In addition, E-specific IL-4 T-cell immune responses were detected by ELISPOT after protein boost and CD8+ specific IFN-γ expression was observed by flow cytometry. Challenge experiments using the heterologous immunization regime revealed protective immunity to homologous and virulent WNV infection.