Trimeric, APC-Targeted Subunit Vaccines Protect Mice against Seasonal and Pandemic Influenza.

Viral subunit vaccines contain the specific antigen deemed most important for development of protective immune responses. Typically, the chosen antigen is a surface protein involved in cellular entry of the virus, and neutralizing antibodies may prevent this. For influenza, hemagglutinin (HA) is thus a preferred antigen. However, the natural trimeric form of HA is often not considered during subunit vaccine development. Here, we have designed a vaccine format that maintains the trimeric HA conformation while targeting antigen toward major histocompatibility complex class II (MHCII) molecules or chemokine receptors on antigen-presenting cells (APC) for enhanced immunogenicity. Results demonstrated that a single DNA vaccination induced strong antibody and T-cell responses in mice. Importantly, a single DNA vaccination also protected mice from lethal challenges with influenza viruses H1N1 and H5N1. To further evaluate the versatility of the format, we developed MHCII-targeted HA from influenza A/California/04/2009(H1N1) as a protein vaccine and benchmarked this against Pandemrix and Flublok. These vaccine formats are different, but similar immune responses obtained with lower vaccine doses indicated that the MHCII-targeted subunit vaccine has an immunogenicity and efficacy that warrants progression to larger animals and humans. IMPORTANCE Subunit vaccines present only selected viral proteins to the immune system and allow for safe and easy production. Here, we have developed a novel vaccine where influenza hemagglutinin is presented in the natural trimeric form and then steered toward antigen-presenting cells for increased immunogenicity. We demonstrate efficient induction of antibodies and T-cell responses, and demonstrate that the vaccine format can protect mice against influenza subtypes H1N1, H5N1, and H7N1.

Antigen bivalency of antigen-presenting cell-targeted vaccines increases B cell responses.

Antibodies are important for vaccine efficacy. Targeting antigens to antigen-presenting cells (APCs) increases antibody levels. Here, we explore the role of antigen valency in MHC class II (MHCII)-targeted vaccines delivered as DNA. We design heterodimeric proteins that carry either two identical (bivalent vaccines), or two different antigens (monovalent vaccines). Bivalent vaccines with two identical influenza hemagglutinins (HA) elicit higher amounts of anti-HA antibodies in mice than monovalent versions with two different HAs. Bivalent vaccines increase the levels of germinal center (GC) B cells and long-lived plasma cells. Only HA-bivalent vaccines completely protect mice against challenge with homologous influenza virus. Similar results are obtained with other antigens by targeting CD11c and Xcr1 on dendritic cells (DCs) or when administering the vaccine as protein with adjuvant. Bivalency probably increases B cell responses by cross-linking BCRs in readily observable DC-B cell synapses. These results are important for generating potent APC-targeted vaccines.

The Highly Productive Thermothelomyces heterothallica C1 Expression System as a Host for Rapid Development of Influenza Vaccines.

(1) Influenza viruses constantly change and evade prior immune responses, forcing seasonal re-vaccinations with updated vaccines. Current FDA-approved vaccine manufacturing technologies are too slow and/or expensive to quickly adapt to mid-season changes in the virus or to the emergence of pandemic strains. Therefore, cost-effective vaccine technologies that can quickly adapt to newly emerged strains are desirable. (2) The filamentous fungal host Thermothelomyces heterothallica C1 (C1, formerly Myceliophthora thermophila) offers a highly efficient and cost-effective alternative to reliably produce immunogens of vaccine quality at large scale. (3) We showed the utility of the C1 system expressing hemagglutinin (HA) and a HA fusion protein from different H1N1 influenza A virus strains. Mice vaccinated with the C1-derived HA proteins elicited anti-HA immune responses similar, or stronger than mice vaccinated with HA products derived from prototypical expression systems. A challenge study demonstrated that vaccinated mice were protected against the aggressive homologous viral challenge. (4) The C1 expression system is proposed as part of a set of protein expression systems for plug-and-play vaccine manufacturing platforms. Upon the emergence of pathogens of concern these platforms could serve as a quick solution for producing enough vaccines for immunizing the world population in a much shorter time and more affordably than is possible with current platforms.

Pandemic Preparedness Against Influenza: DNA Vaccine for Rapid Relief.

The 2009 "swine flu" pandemic outbreak demonstrated the limiting capacity for egg-based vaccines with respect to global vaccine supply within a timely fashion. New vaccine platforms that efficiently can quench pandemic influenza emergences are urgently needed. Since 2009, there has been a profound development of new vaccine platform technologies with respect to prophylactic use in the population, including DNA vaccines. These vaccines are particularly well suited for global pandemic responses as the DNA format is temperature stable and the production process is cheap and rapid. Here, we show that by targeting influenza antigens directly to antigen presenting cells (APC), DNA vaccine efficacy equals that of conventional technologies. A single dose of naked DNA encoding hemagglutinin (HA) from influenza/A/California/2009 (H1N1), linked to a targeting moiety directing the vaccine to major histocompatibility complex class II (MHCII) molecules, raised similar humoral immune responses as the adjuvanted split virion vaccine Pandemrix, widely administered in the 2009 pandemic. Both vaccine formats rapidly induced serum antibodies that could protect mice already 8 days after a single immunization, in contrast to the slower kinetics of a seasonal trivalent inactivated influenza vaccine (TIV). Importantly, the DNA vaccine also elicited cytotoxic T-cell responses that reduced morbidity after vaccination, in contrast to very limited T-cell responses seen after immunization with Pandemrix and TIV. These data demonstrate that DNA vaccines has the potential as a single dose platform vaccine, with rapid protective effects without the need for adjuvant, and confirms the relevance of naked DNA vaccines as candidates for pandemic preparedness.

Integration of T helper and BCR signals governs enhanced plasma cell differentiation of memory B cells by regulation of CD45 phosphatase activity.

Humoral immunity relies on the efficient differentiation of memory B cells (MBCs) into antibody-secreting cells (ASCs). T helper (Th) signals upregulate B cell receptor (BCR) signaling by potentiating Src family kinases through increasing CD45 phosphatase activity (CD45 PA). In this study, we show that high CD45 PA in MBCs enhances BCR signaling and is essential for their effective ASC differentiation. Mechanistically, Th signals upregulate CD45 PA through intensifying the surface binding of a CD45 ligand, Galectin-1. CD45 PA works as a sensor of T cell help and defines high-affinity germinal center (GC) plasma cell (PC) precursors characterized by IRF4 expression in vivo. Increasing T cell help in vitro results in an incremental CD45 PA increase and enhances ASC differentiation by facilitating effective induction of the transcription factors IRF4 and BLIMP1. This study connects Th signals with BCR signaling through Galectin-1-dependent regulation of CD45 PA and provides a mechanism for efficient ASC differentiation of MBCs.

Endocytosis Deficient Murine Xcl1-Fusion Vaccine Enhances Protective Antibody Responses in Mice.

Targeting antigen to surface receptors on dendritic cells (DCs) can improve antibody response against subunit vaccines. We have previously observed that human XCL1-fusion vaccines target murine Xcr1+ DCs without actively inducing endocytosis of the antigen, resulting in enhanced antibody responses in mice. However, the use of foreign chemokines for targeting is undesirable when translating this observation to human or veterinary medicine due to potential cross-reactive responses against the endogenous chemokine. Here we have identified a mutant version of murine Xcl1, labeled Xcl1(Δ1) owing to removal of a conserved valine in position 1 of the mature chemokine, that retains specific binding to Xcr1+ DCs without inducing endocytosis of the receptor. DNA immunization with Xcl1(Δ1) conjugated to influenza hemagglutinin (HA) induced improved antibody responses, with higher end point titers of IgG compared to WT Xcl1-HA. The Xcl1(Δ1) fusion vaccine also resulted in an increased number of HA reactive germinal center B cells with higher avidity toward the antigen, and serum transfer experiments show that Xcl1(Δ1)-HA induced antibody responses provided better protection against influenza infection as compared to WT Xcl1-HA. In summary, our observations indicate that targeting antigen to Xcr1+ DCs in an endocytosis deficient manner enhances antibody responses. This effect was obtained by introducing a single mutation to Xcl1, suggesting our strategy may easily be translated to human or veterinary vaccine settings.

Enhanced germinal center reaction by targeting vaccine antigen to major histocompatibility complex class II molecules.

Enhancing the germinal center (GC) reaction is a prime objective in vaccine development. Targeting of antigen to MHCII on APCs has previously been shown to increase antibody responses, but the underlying mechanism has been unclear. We have here investigated the GC reaction after targeting antigen to MHCII in (i) a defined model with T and B cells of known specificity using adjuvant-free vaccine proteins, and (ii) an infectious disease model using a DNA vaccine. MHCII-targeting enhanced presentation of peptide: MHCII on APCs, and increased the numbers of GC B cells, TFH, and plasma cells. Antibodies appeared earlier and levels were increased. BCR of GC B cells and serum antibodies had increased avidity for antigen. The improved responses required cross-linking of BCR and MHCII in either cis or trans. The enhanced GC reaction induced by MHCII-targeting of antigen has clear implications for design of more efficient subunit vaccines.

Needle-free delivery of DNA: Targeting of hemagglutinin to MHC class II molecules protects rhesus macaques against H1N1 influenza.

Conventional influenza vaccines are hampered by slow and limited production capabilities, whereas DNA vaccines can be rapidly produced for global coverage in the event of an emerging pandemic. However, a drawback of DNA vaccines is their generally low immunogenicity in non-human primates and humans. We have previously demonstrated that targeting of influenza hemagglutinin to human HLA class II molecules can increase antibody responses in larger animals such as ferrets and pigs. Here, we extend these observations by immunizing non-human primates (rhesus macaques) with a DNA vaccine encoding a bivalent fusion protein that targets influenza virus hemagglutinin (HA) to Mamu class II molecules. Such immunization induced neutralizing antibodies and antigen-specific T cells. The DNA was delivered by pain- and needle-free jet injections intradermally. No adverse effects were observed. Most importantly, the immunized rhesus macaques were protected against a challenge with influenza virus.

A DNA Vaccine That Targets Hemagglutinin to Antigen-Presenting Cells Protects Mice against H7 Influenza.

Zoonotic influenza H7 viral infections have a case fatality rate of about 40%. Currently, no or limited human to human spread has occurred, but we may be facing a severe pandemic threat if the virus acquires the ability to transmit between humans. Novel vaccines that can be rapidly produced for global distribution are urgently needed, and DNA vaccines may be the only type of vaccine that allows for the speed necessary to quench an emerging pandemic. Here, we constructed DNA vaccines encoding the hemagglutinin (HA) from influenza A/chicken/Italy/13474/99 (H7N1). In order to increase the efficacy of DNA vaccination, HA was targeted to either major histocompatibility complex class II molecules or chemokine receptors 1, 3, and 5 (CCR1/3/5) that are expressed on antigen-presenting cells (APC). A single DNA vaccination with APC-targeted HA significantly increased antibody levels in sera compared to nontargeted control vaccines. The antibodies were confirmed neutralizing in an H7 pseudotype-based neutralization assay. Furthermore, the APC-targeted vaccines increased the levels of antigen-specific cytotoxic T cells, and a single DNA vaccination could confer protection against a lethal challenge with influenza A/turkey/Italy/3889/1999 (H7N1) in mice. In conclusion, we have developed a vaccine that rapidly could contribute protection against a pandemic threat from avian influenza.IMPORTANCE Highly pathogenic avian influenza H7 constitute a pandemic threat that can cause severe illness and death in infected individuals. Vaccination is the main method of prophylaxis against influenza, but current vaccine strategies fall short in a pandemic situation due to a prolonged production time and insufficient production capabilities. In contrast, a DNA vaccine can be rapidly produced and deployed to prevent the potential escalation of a highly pathogenic influenza pandemic. We here demonstrate that a single DNA delivery of hemagglutinin from an H7 influenza could mediate full protection against a lethal challenge with H7N1 influenza in mice. Vaccine efficacy was contingent on targeting of the secreted vaccine protein to antigen-presenting cells.

Exploring arrays of vertical one-dimensional nanostructures for cellular investigations.

The endeavor of exploiting arrays of vertical one-dimensional (1D) nanostructures (NSs) for cellular applications has recently been experiencing a pronounced surge of activity. The interest is rooted in the intrinsic properties of high-aspect-ratio NSs. With a height comparable to a mammalian cell, and a diameter 100-1000 times smaller, NSs should intuitively reach far into a cell and, due to their small diameter, do so without compromising cell health. Single NSs would thus be expedient for measuring and modifying cell response. Further organization of these structures into arrays can provide up-scaled and detailed spatiotemporal information on cell activity, an achievement that would entail a massive leap forward in disease understanding and drug discovery. Numerous proofs-of-principle published recently have expanded the large toolbox that is currently being established in this rapidly advancing field of research. Encouragingly, despite the diversity of NS platforms and experimental conditions used thus far, general trends and conclusions from combining cells with NSs are beginning to crystallize. This review covers the broad spectrum of NS materials and dimensions used; the observed cellular responses with specific focus on adhesion, morphology, viability, proliferation, and migration; compares the different approaches used in the field to provide NSs with the often crucial cytosolic access; covers the progress toward biological applications; and finally, envisions the future of this technology. By maintaining the impressive rate and quality of recent progress, it is conceivable that the use of vertical 1D NSs may soon be established as a superior choice over other current techniques, with all the further benefits that may entail.

Tuning InAs nanowire density for HEK293 cell viability, adhesion, and morphology: perspectives for nanowire-based biosensors.

Arrays of nanowires (NWs) are currently being established as vehicles for molecule delivery and electrical- and fluorescence-based platforms in the development of biosensors. It is conceivable that NW-based biosensors can be optimized through increased understanding of how the nanotopography influences the interfaced biological material. Using state-of-the-art homogenous NW arrays allow for a systematic investigation of how the broad range of NW densities used by the community influences cells. Here it is demonstrated that indium arsenide NW arrays provide a cell-promoting surface, which affects both cell division and focal adhesion up-regulation. Furthermore, a systematic variation in NW spacing affects both the detailed cell morphology and adhesion properties, where the latter can be predicted based on changes in free-energy states using the proposed theoretical model. As the NW density influences cellular parameters, such as cell size and adhesion tightness, it will be important to take NW density into consideration in the continued development of NW-based platforms for cellular applications, such as molecule delivery and electrical measurements.