key: cord-0785104-87vezzoi
authors: Park, Kyung Soo; Bazzill, Joseph D.; Son, Sejin; Nam, Jutaek; Shin, Seung Won; Ochyl, Lukasz J.; Stuckey, Jeanne A.; Meagher, Jennifer L.; Chang, Louise; Song, Jun; Montefiori, David C.; LaBranche, Celia C.; Smith, Janet L.; Xu, Jie; Moon, James J.
title: Lipid-based vaccine nanoparticles for induction of humoral immune responses against HIV-1 and SARS-CoV-2
date: 2020-12-20
journal: J Control Release
DOI: 10.1016/j.jconrel.2020.12.031
sha: 3342d87ec8db4a226dc64bb8b3cd64fd260799e7
doc_id: 785104
cord_uid: 87vezzoi

The current health crisis of corona virus disease 2019 (COVID-19) highlights the urgent need for vaccine systems that can generate potent and protective immune responses. Protein vaccines are safe, but conventional approaches for protein-based vaccines often fail to elicit potent and long-lasting immune responses. Nanoparticle vaccines designed to co-deliver protein antigens and adjuvants can promote their delivery to antigen-presenting cells and improve immunogenicity. However, it remains challenging to develop vaccine nanoparticles that can preserve and present conformational epitopes of protein antigens for induction of neutralizing antibody responses. Here, we have designed a new lipid-based nanoparticle vaccine platform (NVP) that presents viral proteins (HIV-1 and SARS-CoV-2 antigens) in a conformational manner for induction of antigen-specific antibody responses. We show that NVP was readily taken up by dendritic cells (DCs) and promoted DC maturation and antigen presentation. NVP loaded with BG505.SOSIP.664 (SOSIP) or SARS-CoV-2 receptor-binding domain (RBD) was readily recognized by neutralizing antibodies, indicating the conformational display of antigens on the surfaces of NVP. Rabbits immunized with SOSIP-NVP elicited strong neutralizing antibody responses against HIV-1. Furthermore, mice immunized with RBD-NVP induced robust and long-lasting antibody responses against RBD from SARS-CoV-2. These results suggest that NVP is a promising platform technology for vaccination against infectious pathogens.

As shown during the current COVID-19 pandemic, reliable and efficient vaccine delivery systems are urgently needed for vaccine development against COVID-19 as well as other emerging pathogens [1, 2] . Traditional vaccines based on the live attenuated virus and inactivated virus vaccines are potent activators of the immune system, but they are limited by potential viral reversion and long development and regulatory timeline. On the other hand, protein vaccines with favorable safety profiles have been widely used for prophylactic vaccination against various pathogens, such as hepatitis B and influenza viruses [3] . Yet, protein subunit vaccines often fail to elicit potent and long-lasting immune responses.

These challenges may be addressed by co-administering subunit protein vaccines with potent adjuvants [4] , especially in nanoparticle formulations that allow for their co-delivery to antigen-presenting cells for strong immune activation [5] . There are various nanoparticle vaccine platforms under development, including polymers [6, 7] , gold [8, 9] , silica [10, 11] , and others [12, 13] . In particular, lipid-based nanoparticles are generally considered to have excellent biocompatibility and safety, and they have been used as a vaccine carrier to deliver mRNA [14] , DNA [15] [16] [17] , and peptides [18] .

However, for protein antigens, it remains challenging to preserve their conformational epitopes and achieve robust neutralizing antibody responses using nano-vaccines. In particular, conformational display of antigens in vaccine formulations is crucial as immunogens should present epitopes to which the immune cells recognize, interact, and generate immune responses. Here, we sought to address these challenges by designing a new lipid-based nanoparticle vaccine platform (NVP) that can load and present viral proteins (HIV-1 and SARS-CoV-2 antigens) in a conformational manner for induction of antigen-specific antibody responses.

In particular, previous studies on acquired immunodeficiency syndrome (AIDS) have revealed the presence of broadly neutralizing antibodies in a subset of AIDS patients [19, 20] . As the human immunodeficiency virus-1 (HIV-1) envelope glycoprotein (Env gp) has been identified to induce broadly neutralizing antibodies, various HIV-1 J o u r n a l P r e -p r o o f Journal Pre-proof Env gp immunogens have been developed. Among them, BG505.SOSIP.664 (SOSIP) has emerged as a promising immunogen for inducing neutralizing antibodies against HIV-1 [21] [22] [23] . SOSIP is derived from the BG505 HIV-1 clade A virus, which was isolated from a 6-week-old infant who later developed broadly neutralizing antibodies [23, 24] . The native form of the glycoprotein gp160 is cleaved into gp120 and gp41 subunits during HIV-1 entry into host cells. To utilize gp160 for vaccination purpose, the membrane-associated and cytoplasmic domains were truncated and stabilized by insertion of a disulfide bond (referred to as "SOS") and an Ile/Pro ("IP") substitution at residue 559 (I559P), resulting in an immunogen termed as SOSIP [21] [22] [23] . SOSIP selfassembles into a soluble HIV-1 Env trimer; therefore, nano-vaccine formulation with SOSIP should maintain the structural integrity and neutralizing epitopes of SOSIP.

During the initial COVID-19 outbreak, the structural similarities between SARS-CoV and SARS-CoV-2 were discovered [25] . It was subsequently revealed that spike glycoprotein (S protein) of SARS-CoV-2 was responsible for viral infection via interaction with angiotensin-converting enzyme 2 (ACE2) receptors on human cell membranes and that the antibodies generated against the S protein effectively neutralize viral entry to human cells [26, 27] . The receptor-binding domain (RBD) is the functional domain within the S protein that first engages with ACE2, is considered a prime target for COVID-19 vaccine development, and can be produced as a recombinant antigen to generate directed antibody responses [28, 29] .

It should be noted that both SOSIP and RBD possess tertiary molecular structures through various bonds, including disulfide bond [30, 31] , that are prone to denaturation if placed under harsh condition, e.g., extreme pH, temperature, and physical stress. While we have previously reported lipid-based vaccine nanoparticles that employ thiol-maleimide crosslinking reaction to form nanoparticles [32] [33] [34] [35] , they are not ideal for immunogens held together by disulfide bonds, such as in SOSIP. Therefore, we sought to design a new nano-formulation for loading HIV-1 SOSIP and SARS-CoV-2 RBD while preserving epitopes for inducing antibody responses. We show that pre- These results suggest that NVP is a promising platform technology for vaccination against infectious pathogens. were performed using either unpaired student's t-test or one-way or two-way ANOVA, followed by Tukey's HSD multiple comparison test. Statistical significances are indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

We synthesized a nanoparticle vaccine platform (NVP) by adding viral antigens to pre- (Fig. 1a) . The resulting NVP exhibited a hydrodynamic size of 200-400 nm and a polydispersity index of 0.1-0.3 depending on the antigen added, with slightly negative surface charge as shown by dynamic light scattering (DLS) measurement (Fig. 1b) . 

presentation.

As a model antigen, DQ-labeled ovalbumin (DQ-OVA) was loaded to NVP to examine antigen delivery to and activation of DCs in vitro (Fig. 2a-f ) DQ is a self-quenched dye that emits fluorescence upon degradation after cellular entry [38] . PAGE analysis showed efficient loading (~60%) of DQ-OVA to NVP (Fig. S1) . BMDCs treated for 1 hr with NVP carrying DQ-OVA (DQ-OVA-NVP) exhibited a 2.72-fold increase in the DQ signal, compared with those treated with soluble DQ-OVA and MPLA mixture, as confirmed by flow cytometry (Fig. 2a) . A similar trend was observed over 24 hr (Fig. 2b) .

As shown by confocal microscopy, BMDCs treated with DQ-OVA-NVP for 4 hr showed significantly higher DQ signal within the cytosol, with a high level of co-localization with lysosomes (Fig. 2g) . In addition, DQ-OVA-NVP induced robust DC maturation, as shown by the up-regulation of CD80 co-stimulatory marker on DCs within 1 hr of incubation (Fig. 2c) and throughout 24 hr time window (Fig. 2d) . Moreover, we examined antigen presentation on DCs by flow cytometry assay after staining DCs with a monoclonal antibody specific to an immunodominant OVA epitope (SIINFEKL) loaded in major histocompatibility complex-I (MHC-I) molecule. NVP-mediated DQ-OVA delivery led to significantly greater antigen presentation on DCs (Fig. 2e-f) . Taken together, these results show that NVP significantly increases DC uptake of vaccines, leading to improved DC activation and antigen presentation. 

Next, we investigated NVP-mediated lymph node trafficking of antigen using a model antigen, Alexa Fluor 647 (AF647)-labeled ovalbumin (AF647OVA). Mice were given tailbase subcutaneous injection of PBS, soluble AF647OVA + MPLA, or AF647OVAloaded NVP (OVA-MPLA-NVP), followed by flow cytometry or ELISA analyses (Fig. 3a) .

After 48 hr of vaccination, draining inguinal lymph nodes were visualized by IVIS fluorescence imaging. Mice administered with OVA-MPLA-NVP had significantly stronger AF647 signal in lymph nodes, compared with those treated with the soluble formulation (Fig. 3b) . Flow cytometric analysis showed that CD11c+ DCs in lymph nodes from the OVA-MPLA-NVP group exhibited signs of maturation, as shown by CD80 and CD86 staining (Fig. 3c) . Interestingly, among CD80+ and CD86+ DCs, the OVA-MPLA-NVP group had significantly higher mean fluorescence intensity of AF647OVA, compared with the soluble vaccine group (Fig. 3d) , showing robust DCtargeted delivery of antigen by NVP. Lastly, the serum levels of proinflammatory cytokines, IL-6 and IL-12p40, were measured using ELISA. Serum concentrations of IL-6 and IL-12 were elevated at 4 hr and 8 hr post injection, respectively, for the soluble vaccine group (Fig. 3e) , whereas there was no spike of either cytokines in the NVP group. Taken together, these results showed that NVP provides an efficient and safe platform for antigen delivery to antigen-presenting cells in lymph nodes.

Journal Pre-proof 

BG505.SOSIP.664 (SOSIP) is a recombinant HIV-1 envelope glycoprotein derived from the BG505 clade A virus. SOSIP is held together by a disulfide bond and selfassembles into a soluble HIV-1 Env trimer (Fig. 4a) . SOSIP is a promising immunogen for HIV-1 vaccine development, as shown by prior pre-clinical studies reporting SOSIPmediated induction of neutralizing antibodies against HIV-1 [21, 22, 39] . Here, we prepared NVP carrying SOSIP antigen and examined its efficacy to induce neutralizing antibody response against HIV-1. Using the procedure described above, we loaded SOSIP into NVP (SOSIP-NVP), which exhibited a hydrodynamic size of ~330 nm, as determined by DLS analysis (Fig. 1b) . PAGE-based quantification indicated a ~25% loading efficiency of SOSIP in NVP (Fig. 4b) . Notably, it is crucial to maintain the conformational epitopes and trimeric structure of HIV-1 Env for the induction of broadly neutralizing antibody responses [40] . Therefore, we examined whether SOSIP-NVP preserves the structure and epitopes of SOSIP during the vaccine formulation. Our nonreducing PAGE analysis performed on SOSIP retrieved from SOSIP-NVP indicated that SOSIP-NVP maintained the disulfide bond in SOSIP without disruption during the loading process (Fig. 4b) . SOSIP also appeared in the high molecular weight area in the PAGE gel, which may have been due to complexation with PEI and incomplete retrieval process from SOSIP-NVP. In addition, the preservation of quaternary structure of SOSIP trimer after NVP loading was examined by blue-native PAGE. Interestingly, application of a significant physical stress (e.g. tip sonication) while SOSIP trimer is present in solution induced dissociation of the trimer into monomer and dimer, demonstrating the delicate binding force between the subunits (Fig. 4c, 3 rd lane) . Thus, we modified the SOSIP-NVP preparation by adding SOSIP to the reaction mixture after any physical stresses were taken place, which resulted in the preservation of intact quaternary structure after SOSIP-NVP formulation (Fig. 4c, 5 th lane) . Nevertheless, these results indicated that SOSIP was effectively loaded into NVP.

To further examine whether SOSIP was displayed on the surface of NVP with its epitopes remaining intact, we performed immunofluorescence directly on SOSIP-NVP.

For this, we employed Env-specific human antibodies, PGV04 and b6, which recognize the CD4 binding site of Env. PGV04 and b6 are HIV-1 broadly neutralizing antibody and non-neutralizing antibody, respectively. PGV04 and b6 were incubated with SOSIP-NVP, followed by washing and another round of incubation with fluorophore-tagged antihuman IgG antibody. Then fluorescence signal on SOSIP-NVP was quantified to assess antibody binding (Fig. 4d, e) . SOSIP-NVP was readily recognized and bound by PGV04, a broadly neutralizing antibody, on a whole population level, as shown by a plate-based fluorescence measurement (Fig. 4d) . SOSIP-NVP was also bound by b6, a nonneutralizing antibody, but to a lesser extent than PGV04. We recently reported that antibody-binding on nanoparticles could be quantified on an individual nanoparticlebasis using "NanoFACS" [33] . Using NanoFACS, we confirmed that PGV04 was bound to individual SOSIP-NVPs (Fig. 4e) Loading of SOSIP into NVP confirmed by non-reducing PAGE, followed by silver staining. c) Blue native PAGE showing intact SOSIP trimer before or after physical disruption as well as after loading in NVP using an optimized formulation condition. d-e) To examine SOSIP display on NVP, human anti-SOSIP antibodies, b6 and PGV04, were incubated with SOSIP-NVP and PE-labeled anti-human IgG antibody, followed by quantification of antibody binding by d) plate-based fluorescence measurement and e) individual nanoparticle-based flow cytometry. Data are presented as mean ± SEM. *p < 0.05, ***p < 0.001, analyzed by one-way ANOVA, followed by Tukey's HSD multiple comparison post hoc test.

We performed immunization studies with SOSIP-NVP and examined their potency to generate neutralizing antibody response in rabbits. White New Zealand rabbits were immunized on day 0 with 30 µg SOSIP and 50 µg MPLA, followed by two boost immunizations on days 28 and 84, each with 12.4 µg SOSIP and 20.6 µg MPLA (Fig.   5a) . SOSIP and MPLA were administered subcutaneously either in SOSIP-NVP or soluble formulations. Sera samples collected on days 28, 56, 105 and 169 were assessed for neutralization against HIV-1 viral entry to TZM-bl cells using heterologous tier 1A virus (MW965.26, clade C) and autologous tier 2 virus (BG505/T332N) [41, 42] .

On Day 56 and more noticeably on Day 105, immune sera from the SOSIP-NVP vaccine group showed strong neutralizing activity against heterologous titer 1A MW965.26 virus (Fig. 5b) , with a trend for increasing neutralizing activity compared with the soluble vaccine group. Day 169 immune sera also showed neutralizing activity, although dampened compared with day 105, against MW965.26 (Fig. 5b) . As (Fig. 5c) . Moreover, there was also a trend for higher neutralizing antibody titers against autologous BG505/T332N up to day 169 ( Fig. 5c) . As it has been challenging to produce vaccines capable of neutralizing against a tier 2 virus, even an autologous one such as BG505/T332N virus, these results show the promise of SOSIP-NVP for vaccination against HIV-1.

J o u r n a l P r e -p r o o f 

Motivated by the promising results of SOSIP-NVP, we sought to apply the NVP technology for COVID-19 vaccine development. According to previous studies, the spike protein (S protein) of SARS-CoV-2 is responsible for the interaction with ACE2 receptors on the human cells which leads to viral infection. Inducing antibody response against S protein therefore is an effective strategy to neutralize SARS-CoV-2 [26, 27] .

The receptor-binding domain (RBD) is the functional region within the S protein that engages ACE2, and has been suggested as a great target for vaccines against SARS-CoV-2 [28, 29] . In addition, our analysis on genetic sequence comparison between SARS-CoV-2 variants reported on National Center for Biotechnology Information (NCBI) J o u r n a l P r e -p r o o f as of August 2020 and the original SARS-CoV-2 that appeared in Wuhan in 2019 indicated high conservation of RBD genetic sequence, compared with other genetic regions ( Fig. 6a-b) , thus highlighting RBD as a promising target for COVID-19 vaccine development.

We synthesized RBD-loaded NVP as described above. Loading of RBD in NVP was quantified by PAGE analysis, which showed ~21% loading efficiency (Fig. 6c) . The resulting RBD-loaded NVP (RBD-NVP) had a hydrodynamic size of ~240 nm as measured by DLS (Fig. 1b) . Surface-display of RBD on RBD-NVP was examined by direct immunofluorescence as described. RBD-NVP was incubated with human RBD neutralizing antibodies, followed by washing and addition of AF488-labeled anti-human IgG secondary antibody. RBD-NVP exhibited a significantly higher fluorescence signal, compared with blank NVP control (Fig. 6d) , indicating the proper display of RBD and preservation of epitopes in RBD-NVP.

J o u r n a l P r e -p r o o f Figure 6 . Genomic deviation of SARS-CoV-2 by coding region and characterization of RBD-NVP. (a) Spike protein is the most genetically conserved region within the genetic sequence of SARS-CoV-2, based on variants appearing with near 100% similarity to the original SARS-CoV-2 in this region. (b) RBD of SARS-CoV-2 variants has the highest sequence similarity to that of the original SASR-CoV-2 with the smallest deviation, compared with other domains. (c) RBD loading in NVP was confirmed by SDS-PAGE analysis. (d) RBD display on NVP surface was assessed by incubation with human anti-SARS-CoV-2 neutralizing antibody, followed by incubation with Alexa Fluor 488-labeled anti-human IgG1 Fc secondary antibody. Antibody bound to NVP was quantified by fluorometry. Data are presented as mean ± SEM. ***p < 0.001, ****p < 0.0001, analyzed by one-way ANOVA, followed by Tukey's HSD multiple comparison post hoc test.

Lastly, we examined the potency of RBD-NVP to generate anti-RBD antibody response in mice. BALB/c mice were vaccinated three times with 2 weeks interval between each injection (days 0, 14, and 28) using 0.5 µg of RBD and 1 µg of MPLA either in NVP or soluble formulation (Fig. 7a) . Sera samples were collected on days 28, 42, and 70 and assessed for RBD-specific serum IgG, IgG 1 , and IgG 2a titers. RBD-NVP generated significantly higher RBD-specific antibody titers, compared with RBD + MPLA soluble vaccine ( Fig. 7b-d) . Specifically, by day 42 (2 weeks after 3 rd vaccination), RBD-NVP elicited 55-fold, 17-fold, and 284-fold higher RBD-specific IgG, IgG 1 , and IgG 2a titers, respectively, compared with the soluble vaccine ( Fig. 7b-d) . By day 70 (6 weeks after 3 rd vaccination), mice immunized with RBD-NVP still maintained 30-fold, 13-fold, and 671-fold higher RBD-specific IgG, IgG 1 , and IgG 2a titers, respectively, compared with the soluble vaccine group (Fig. 7b-d) . While the examination of functionality and neutralizing activities of these antibodies are beyond the scope of our current studies, these initial results indicated that RBD-NVP induced robust, long-lasting, Th1/Th2balanced antibody responses against RBD.

Overall, we have developed NVP for the delivery of protein antigens and demonstrated the versatility of NVP for protein-based vaccination against infectious pathogens. Protein antigens incorporated into NVP maintained the configuration of the epitopes, as shown by the recognition and binding of neutralizing antibodies on the J o u r n a l P r e -p r o o f surfaces of antigen-displaying NVP. NVP was readily taken up by DCs in vitro, leading to greater DC activation and antigen presentation, compared with soluble vaccine formulation. We have successfully prepared NVP carrying SOSIP derived from HIV-1 and RBD derived from SARS-CoV-2. Animals immunized with NVP generated strong antigen-specific antibody responses. While these initial proof-of-concept studies have shown the promise of NVP, more studies are warranted to delineate the immunological mechanisms of action and to assess protective immunity against viral challenge (e.g., HIV-1 or SHIV challenge in non-human primates; and SARS-CoV-2 challenge in mice engineered to express human ACE2).

BALB/c mice were vaccinated three times, with 2 weeks intervals between each injection. Blood was sampled on the indicated days. RBD-NVP significantly increased serum antibody titers of RBD-specific (b) IgG, (c) IgG1, and (d) IgG2a, compared to the soluble vaccine. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001, analyzed by two-way ANOVA, followed by Tukey's HSD multiple comparison post hoc test.

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Graphical abstract

A nanoparticle vaccine platform that preserves conformational epitopes of protein antigens elicits robust humoral immune responses against HIV-1 and SARS-CoV-2

There is no conflict of interest to declare.