Vaccine-Mediated Induction of an Ebolavirus Cross-Species Antibody Binding to Conserved Epitopes on the Glycoprotein Heptad Repeat 2/Membrane-Proximal External Junction.

The membrane-proximal external regions (MPER) of the human immunodeficiency virus envelope glycoprotein (GP) generate broadly reactive antibody responses and are the focus of vaccine development efforts. The conservation of amino acids within filovirus GP heptad repeat region (HR)2/MPER suggests that it may also represent a target for a pan-filovirus vaccine. We immunized a cynomolgus macaque against Ebola virus (EBOV) using a deoxyribonucleic acid/adenovirus 5 prime/boost strategy, sequenced memory B-cell receptors, and tested the antibodies for functional activity against EBOV GP. Antibody ma-C10 bound to GP with an affinity of 48 nM and was capable of inducing antibody-dependent cellular cytotoxicity. Three-dimensional reconstruction of single-particle, negative-stained, electron microscopy showed that ma-C10 bound to the HR2/MPER, and enzyme-linked immunosorbent assay reveals it binds to residues 621-631. More importantly, ma-C10 was found to bind to the GP of the 3 most clinically relevant Ebolavirus species, suggesting that a cross-species immunogen strategy targeting the residues in this region may be a feasible approach for producing a pan-filovirus vaccine.

Functional interrogation and mining of natively paired human VH:VL antibody repertoires.

We present a technology to screen millions of B cells for natively paired human antibody repertoires. Libraries of natively paired, variable region heavy and light (VH:VL) amplicons are expressed in a yeast display platform that is optimized for human Fab surface expression. Using our method we identify HIV-1 broadly neutralizing antibodies (bNAbs) from an HIV-1 slow progressor and high-affinity neutralizing antibodies against Ebola virus glycoprotein and influenza hemagglutinin.

The improved antibody response against HIV-1 after a vaccination based on intrastructural help is complemented by functional CD8+ T cell responses.

Despite more than three decades of intense research, a prophylactic HIV-1 vaccine remains elusive. Four vaccine modalities have been evaluated in clinical efficacy studies, but only one demonstrated at least modest efficacy, which correlated with polyfunctional antibody responses to the HIV surface protein Env. To be most effective, a HIV-1 vaccine probably has to induce both, functional antibody and CD8(+) T cell responses. We therefore analyzed DNA/DNA and DNA/virus-like particle (VLP) regimens for their ability to induce humoral and cellular immune responses. Here, DNA vaccination of mice induced strong CD8(+) responses against Env and Gag. However, the humoral response to Env was dominated by IgG1, a subclass known for its low functionality. In contrast, priming only with the Gag-encoding plasmid followed by a boost with VLPs consisting of Gag and Env improved the quality of the anti-Env antibody response via intrastructural help (ISH) provided by Gag-specific T cells to Env-specific B cells. Furthermore, the Gag-specific CD8(+) T cells induced by the DNA prime immunization could still protect from a lethal infection with recombinant vaccinia virus encoding HIV Gag. Therefore, this immunization regimen represents a promising approach to combine functional antibody responses toward HIV Env with strong CD8(+) responses controlling early viral replication.

Enhancing the Quality of Antibodies to HIV-1 Envelope by GagPol-Specific Th Cells.

The importance of Fc-dependent effector functions of Abs induced by vaccination is increasingly recognized. However, vaccination of mice against HIV envelope (Env) induced a skewed Th cell response leading to Env-specific Abs with reduced effector function. To overcome this bias, GagPol-specific Th cells were harnessed to provide intrastructural help for Env-specific B cells after immunization with virus-like particles containing GagPol and Env. This led to a balanced Env-specific humoral immune response with a more inflammatory Fc glycan profile. The increased quality in the Ab response against Env was confirmed by FcγR activation assays. Because the Env-specific Th cell response was also biased in human vaccinees, intrastructural help is an attractive novel approach to increase the efficacy of prophylactic HIV Env-based vaccines and may also be applicable to other particulate vaccines.

DNA vaccines encoding DEC205-targeted antigens: immunity or tolerance?

Targeting of antigens to the endocytic uptake receptor DEC205 resulted in enhanced antigen presentation by dendritic cells (DCs). In combination with adjuvants for DC maturation, proteins coupled to an antibody against DEC205 induced strong pathogen-specific immune responses, whereas without additional adjuvant tolerance could be induced. As less is known about DNA vaccines encoding DEC205-targeted antigens, we explored the immunogenicity and efficacy of a dendritic cell-targeted DNA vaccine against influenza A virus (IAV) delivered by electroporation. Although coupling of haemagglutinin to a single-chain antibody against DEC205 enhanced antigen presentation on MHC class II and activation of T-cell receptor-transgenic CD4 T cells, the T-cell responses induced by the targeted DNA vaccine in wild-type BALB/c mice were significantly reduced compared with DNA encoding non-targeted antigens. Consistently, these mice were less protected against an IAV infection. Adoptive transfer experiments were performed to assess the fate of the antigen-specific T cells in animals vaccinated with DNA encoding DEC205-targeted antigens. By this, we could exclude the general deletion of antigen-specific T cells as cause for the reduced efficacy, but observed a local expansion of antigen-specific regulatory T cells, which could suppress the activation of effector cells. In conclusion, DNA vaccines encoding DEC205-targeted antigens induce peripheral tolerance rather than immunity in our study. Finally, we evaluated our DNA vaccines as prophylactic or therapeutic treatment in an allergen-induced asthma mouse model.

Divergence of primary cognate B- and T-cell proliferative responses to subcutaneous and intravenous immunization with virus-like particles.

A major advantage of virus-like particle (VLP) vaccines against HIV is their structural identity to wild-type viruses, ensuring that antigen-specific B-cells encounter the envelope protein in its natural conformation. For the induction of affinity-matured antibodies, the B-cells must also obtain help from T-cells that are restricted by linear epitopes. Using B- and T-cell transgenic mouse models, we compared the efficacy of modified HIV-VLPs delivered by subcutaneous and intravenous immunization to stimulate primary B- and T-cell proliferative responses in different lymphoid organs. VLPs containing an influenza virus hemagglutinin epitope within the HIV-Gag protein induced comparable primary cognate T-cell proliferative responses in the draining lymph node and the spleen, irrespective of the delivery route. In contrast, after subcutaneous immunization with HIV-Gag VLPs containing hen egg lysozyme (HEL) on their surface, the proliferative response of transgenic HEL-specific B-cells was restricted to the draining lymph nodes, while intravenous VLP immunization primarily induced a B-cell proliferative response in the spleen. In vitro co-culture experiments further revealed that the presentation of VLP-associated surface antigens by dendritic cells to cognate B-cells is inefficient. This is consistent with a direct triggering of the B-cell proliferative response by the VLPs and suggests that HIV VLPs may indeed be suitable to directly promote the expansion of B-cells specific for conformational epitopes that are unique to functionally-active Env spikes on the virion. Further investigations are warranted to explore potential differences in the quality and protective potency of HIV-specific antibody responses induced by the two routes.

Protective efficacy and immunogenicity of a combinatory DNA vaccine against Influenza A Virus and the Respiratory Syncytial Virus.

The Respiratory Syncytial Virus (RSV) and Influenza A Virus (IAV) are both two major causative agents of severe respiratory tract infections in humans leading to hospitalization and thousands of deaths each year. In this study, we evaluated the immunogenicity and efficacy of a combinatory DNA vaccine in comparison to the single component vaccines against both diseases in a mouse model. Intramuscular electroporation with plasmids expressing the hemagglutinin (HA) of IAV and the F protein of RSV induced strong humoral immune responses regardless if they were delivered in combination or alone. In consequence, high neutralizing antibody titers were detected, which conferred protection against a lethal challenge with IAV. Furthermore, the viral load in the lungs after a RSV infection could be dramatically reduced in vaccinated mice. Concurrently, substantial amounts of antigen-specific, polyfunctional CD8⁺ T-cells were measured after vaccination. Interestingly, the cellular response to the hemagglutinin was significantly reduced in the presence of the RSV-F encoding plasmid, but not vice versa. Although these results indicate a suppressive effect of the RSV-F protein, the protective efficacy of the combinatory vaccine was comparable to the efficacy of both single-component vaccines. In conclusion, the novel combinatory vaccine against RSV and IAV may have great potential to reduce the rate of severe respiratory tract infections in humans without increasing the number of necessary vaccinations.

Comparison of polystyrene nanoparticles and UV-inactivated antigen-displaying adenovirus for vaccine delivery in mice.

BACKGROUND: Inert nanoparticles are attracting attention as carriers for protein-based vaccines. Here we evaluate the immunogenicity of the model antigen ovalbumin delivered on polystyrene particles and directly compare particulate delivery with adenovirus-based immunization. FINDINGS: Mice were vaccinated with soluble ovalbumin, ovalbumin-coated polystyrene particles of different sizes, or an adenovirus-based expression-display vector that encodes and displays a pIX-ovalbumin fusion protein. Antibody responses were clearly higher when ovalbumin was administered on polystyrene particles compared to soluble protein administration, regardless of the particle size. Compared to adenovirus-based immunization, antibody levels were lower if an equivalent amount of protein was delivered, and no cellular immune response was detectable. CONCLUSIONS: We demonstrate in a side-by-side comparison that inert nanoparticles allow for the reduction of the administered antigen amount compared to immunization with soluble protein and induce strongly enhanced antibody responses, but responses are lower compared to adenovirus-based immunization.