Interplay of diverse adjuvants and nanoparticle presentation of native-like HIV-1 envelope trimers

The immunogenicity of HIV-1 envelope (Env) trimers is generally poor. We used the clinically relevant ConM SOSIP trimer to compare the ability of different adjuvants (squalene emulsion, ISCOMATRIX, GLA-LSQ, and MPLA liposomes) to support neutralizing antibody (NAb) responses in rabbits. The trimers were administered as free proteins or on nanoparticles. The rank order for the adjuvants was ISCOMATRIX > SE > GLA-LSQ ~ MPLA liposomes > no adjuvant. Stronger NAb responses were elicited when the ConM SOSIP trimers were presented on ferritin nanoparticles. We also found that the GLA-LSQ adjuvant induced an unexpectedly strong antibody response to the ferritin core of the nanoparticles. This "off-target" effect may have compromised its ability to induce the more desired antitrimer antibodies. In summary, both adjuvants and nanoparticle display can improve the magnitude of the antibody response to SOSIP trimers but the best combination of trimer presentation and adjuvant can only be identified experimentally

Investigation of particulate HIV-1 Env vaccine candidates using Zera® and SpyTag/SpyCatcher technologies

The HIV-1 envelope glycoprotein (Env) is the primary focus of prophylactic HIV vaccine development. However, the unusually low density of Env spikes on the virion (≈14 spikes/virion) is unfavourable for eliciting high titre, long-lasting antibody responses. It is possible that increasing the Env spike density of particulate vaccine candidates generated by protein body formation or via the display of Env on nanoparticles could improve the induction of long-lasting neutralising antibodies (NAbs). For this thesis, two different nanoparticle approaches were therefore investigated. The HIV-1 Env sequence used for both approaches was derived from the superinfecting subtype C CAP256 virus. This was truncated to remove the transmembrane domain, and engineered to contain a flexible linker (FL) in place of the furin cleavage site and an I559P mutation to generate soluble, stable and cleavageindependent gp140 proteins. The first approach investigated the impact of genetically fusing a 27 kDa proline-cysteine-rich domain of the ɣ-zein maize seed storage protein - Zera® - to either the N- or C-terminus of CAP256 gp140. Fusion of Zera® to a protein of interest can promote the self-assembly of large protein bodies (PBs) containing the protein of interest, thereby improving yields of the recombinant protein and enabling easy isolation using gradient ultracentrifugation. The purification of Zera-induced Env PBs from infiltrated Nicotiana benthamiana plants was not optimal. Consequently, the generation of Zera®-induced gp140 protein bodies was evaluated in a mammalian expression system. Stable HEK293 cell lines expressing Zera®-gp140 or gp140-Zera® were generated. A mixture of small PB-like structures was observed in cells expressing gp140-Zera®. However, no PB-like structures were seen in cells expressing Zera®-gp140. The immunogenicity of Zera®-gp140 and gp140-Zera® was evaluated by in rabbits. Binding and Tier 1A neutralising serum titres were higher for gp140-Zera® than for Zera®-gp140. Neither gp140-Zera® nor Zera®-gp140-specific sera neutralised a Tier 1B pseudovirus or the autologous Tier 2 CAP256SU pseudovirus, suggesting that Zera® might have compromised the structure of the Zera®-tagged gp140 proteins. The second approach investigated the two-component SpyCatcher/SpyTag technology. The stable HEK293 cell line expressing CAP256 gp140-SpyTag (gp140-ST) was generated, and trimers were purified to homogeneity using gel filtration. SpyCatcher (SC)-AP205 VLPs were produced in E. coli and purified by ultracentrifugation. The gp140-ST trimers and the SCAP205 VLPs were mixed in varying molar ratios to generate VLPs displaying the glycoprotein (AP205-gp140-ST particles). SDS-PAGE, dynamic light scattering and negative stain electron microscopy indicated that gp140-ST was successfully bound to the VLPs, although not all potential binding sites were occupied. The immunogenicity of the coupled VLPs was evaluated in a pilot study in rabbits. One group was injected four times with coupled VLPs. The second group was primed with DNA vaccines expressing Env and a mosaic Gag, followed by modified vaccinia Ankara expressing the same antigens and then boosted twice with coupled VLPs. Encouragingly, gp140-ST displayed on SC-AP205 VLPs was an effective boost to heterologously primed rabbits, leading to induction of autologous Tier 2 neutralising antibodies in 2/5 rabbits. These results demonstrate that careful selection of a geometrically-suitable nanoparticle scaffold to achieve a high-density display of HIV-1 envelope trimers is an important consideration and that this could improve the effect of nanoparticle-displayed gp140

Formulation of Lipid Nanoparticles with Viral Subunit Antigens for Vaccination

Vaccination provides significant advantages over post-infection treatment such as long-lasting protection, prevention of co-morbidities, and a reduction in the dissemination of pathogens. While vaccination has tempered many once virulent pathogens, others remain without effective vaccines. Moreover, the emergence of previously unknown or isolated pathogens is presenting a significant threat to human health. Overpopulation, increased urbanization, and international travel provide continuous sources of naïve hosts, permitting the persistence and spread of pathogens along with an increased potential of pandemics. Here, three projects are presented describing the development and characterization of viral subunit loaded vaccine nanoparticles for the generation of protective humoral immune responses against hepatitis C virus, Ebola virus, and human immunodeficiency virus. In the first project, lipid-based nanoparticles, called interbilayer-crosslinked multilamellar vesicles (ICMVs), were produced with hepatitis C virus (HCV) recombinant antigens E2.661 or E2c.661, displayed average antigen loading efficiencies were 54% and 50%, respectively, and average nanoparticle dimeters between 115-132 nm. The preservation of surface displayed antigens was confirmed by indirect immunofluorescence staining with antigen-specific antibodies, and in vivo vaccination of mice with ICMV formulations generated ~10-fold higher antigen-specific serum IgG titers compared with control vaccine formulations. Immune sera were tested for their neutralization capacities by an in vitro assay, and both ICMV formulations exhibited neutralization of autologous and heterologous HCV virus like particles, with E2c.661 ICMVs displaying a balanced neutralization profile compared to E2.661 ICMVs, indicating E2c.661 as a candidate antigen for a broadly effective vaccine formulation. In a second application, recombinant Ebola envelope glycoprotein (rGP) was formulated with ICMVs or a variant (NTA ICMVs) for concerted display of rGP on ICMV surfaces. Loading efficiencies varied between formulations (15 and 33%), with the addition of NTA approximately doubling rGP loading. The large rGP complex and epitope conformations were preserved throughout nanoparticle synthesis, and both formulations displayed distinct antibody binding profiles. Regardless of the surface antigen display, both nanoparticle formulations generated marked titers of class switched antigen-specific antibodies in mice after vaccination compared to the vehicle or rGP control groups. Four weeks after immunization, mice were challenged with a lethal dose of murine adapted Ebola virus and 100% survival was observed for mice vaccinated with either ICMV formulation as well as the adjuvanted control formulation. While these data demonstrated short-term protection in three of the tested groups, further research is needed to evaluate long-term protection and the epitope specificity of the generated antibodies. Lastly, a new ICMV nanoparticle design was developed for formulation with the recombinant human immunodeficiency virus envelope glycoprotein (SOSIP). The new nanoparticle, called ICMV-NHS, display ~25% loading efficiency of SOSIP, and a mean diameter of ~300 nm. Preliminary studies indicate preservation of the SOSIP protein complex and conformational epitopes, which are necessary to produce protective and broadly neutralizing humoral responses. However, further optimization and characterization of the nanoparticle are needed to enhance antigen loading and evaluate antigen display prior to in vivo immunogenicity studies. The data reported here highlights the complexity of formulating subunit-loaded vaccine nanoparticles. Many factors including antigen design, display, and antigen-nanoparticle interfaces are important considerations and can contribute significantly to strength and specificity of the generated immune response. To bridge this gap of knowledge, in-depth characterization of nanoparticles, like those reported here, can aid in elucidating and correlating in vitro properties of vaccine nanoparticles with in vivo performance.PHDPharmaceutical SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/147695/1/jbazzill_1.pd

The emergence of nanovaccines as a new paradigm in virological vaccinology: a review

Vaccination has made an enormous contribution to global health. Treatment resistance for infectious diseases is growing quickly, and chemotherapeutic toxicity in cancer means that vaccines must be made right away to save humanity. But subunit vaccinations alone don’t give enough strong and long-lasting protection against infections that can kill. Nanoparticle (NP)-based delivery vehicles, such as dendrimers, liposomes, micelles, virosomes, nanogels, and microemulsions, offer interesting ways to get around the problems with traditional vaccine adjuvants. The nanovaccines (50–250 nm in size) are most efficient in terms of tissue targeting, staying in the bloodstream for a long time. Nanovaccines can improve antigen presentation, targeted delivery, stimulation of the body’s innate immune system, and a strong T-cell response without putting people at risk. This can help fight infectious diseases and cancers. Also, nanovaccines can be very helpful for making cancer treatments that use immunotherapy. So, this review highlights the various types of NPs used in the techniques that have worked in the new paradigm in viral vaccinology for infectious diseases. It gives a full rundown of the current NP-based vaccines, their potential as adjuvants, and the ways they can be delivered to cells. In the future, the best nanovaccines will try to be more logically designed, have more antigens in them, be fully functionalized, and be given to the right people

Virus-like particles and nanoparticles for vaccine development against HCMV

Human cytomegalovirus (HCMV) infects more than 70% of the human population worldwide. HCMV is responsible for high morbidity and mortality in immunocompromised patients and remains the leading viral cause of congenital birth defects. Despite considerable efforts in vaccine and therapeutic development, HCMV infection still represents an unmet clinical need and a life-threatening disease in immunocompromised individuals and newborns. Immune repertoire interrogation of HCMV seropositive patients allowed the identification of several potential antigens for vaccine design. However, recent HCMV vaccine clinical trials did not lead to a satisfactory outcome in term of efficacy. Therefore, combining antigens with orthogonal technologies to further increase the induction of neutralizing antibodies could improve the likelihood of a vaccine to reach protective efficacy in humans. Indeed, presentation of multiple copies of an antigen in a repetitive array is known to drive a more robust humoral immune response than its soluble counterpart. Virus-like particles (VLPs) and nanoparticles (NPs) are powerful platforms for multivalent antigen presentation. Several self-assembling proteins have been successfully used as scaffolds to present complex glycoprotein antigens on their surface. In this review, we describe some key aspects of the immune response to HCMV and discuss the scaffolds that were successfully used to increase vaccine efficacy against viruses with unmet medical need

An engineered HIV-1 Gag-based VLP displaying high antigen density induces strong antibody-dependent functional immune responses

Antigen display on the surface of Virus-Like Particles (VLPs) improves immunogenicity compared to soluble proteins. We hypothesised that immune responses can be further improved by increasing the antigen density on the surface of VLPs. In this work, we report an HIV-1 Gag-based VLP platform engineered to maximise the presence of antigen on the VLP surface. An HIV-1 gp41-derived protein (Min), including the C-terminal part of gp41 and the transmembrane domain, was fused to HIV-1 Gag. This resulted in high-density MinGag-VLPs. These VLPs demonstrated to be highly immunogenic in animal models using either a homologous (VLP) or heterologous (DNA/VLP) vaccination regimen, with the latter yielding 10-fold higher anti-Gag and anti-Min antibody titres. Despite these strong humoral responses, immunisation with MinGag-VLPs did not induce neutralising antibodies. Nevertheless, antibodies were predominantly of an IgG2b/IgG2c profile and could efficiently bind CD16-2. Furthermore, we demonstrated that MinGag-VLP vaccination could mediate a functional effect and halt the progression of a Min-expressing tumour cell line in an in vivo mouse model

Nanoparticle-Based Vaccines against Zoonotic Viruses: A Review

Vaccines are the most promising tools for maintaining public health. Most emerging human infectious diseases are caused by viruses originating from an animal reservoir via zoonotic transmission. Therefore, zoonotic virus spillover and spread in humans have become global health threats. Nanoparticle-based vaccines are ideal for antigen delivery, as adjuvants, and as viral structure mimics. Nanoparticles benefit vaccine design and are utilized to protect the antigen cargo, and increase the immunogenicity and efficacy. Therefore, nanoparticle vaccines are a novel method of immunization by which optimal immune responses are elicited. Herein we review current approaches in the development of nanoparticle vaccines and highlight the role of nanoparticle vaccines against zoonotic viral diseases