Elsevier

Vaccine

Volume 34, Issue 48, 21 November 2016, Pages 5886-5894
Vaccine

Toll-like receptor 3 adjuvant in combination with virus-like particles elicit a humoral response against HIV

https://doi.org/10.1016/j.vaccine.2016.10.036Get rights and content

Highlights

Abstract

Human Immunodeficiency Virus (HIV) Virus-Like Particles (VLPs) composed of HIVIIIB Gag and HIVBaL gp120/gp41 envelope are a pseudovirion vaccine capable of presenting antigens in their native conformations. To enhance the immunogenicity of the HIV Env antigen, VLPs were coupled to VesiVax Conjugatable Adjuvant Lipid Vesicles (CALV) containing one of four toll-like-receptor (TLR) ligands, each activating a receptor with distinct cellular localization and downstream pathways. C57BL/6 mice were vaccinated by intranasal prime followed by two sub-cheek boosts and their sera immunoglobulin and neutralizing potency were measured over a duration of 3 months after vaccination. PBS control, VLPs alone, CALV + VLPs, and VLPs complexed with CALV and ligands for TLR2 (PAM3CAG), TLR3 (dsRNA), TLR4 (MPLA), or TLR7/8 (resiquimod) were evaluated based on antibody titer, IgG1 and IgG2c class switching, germinal center formation, T follicular cells and potency of neutralizing antibodies. Consistently, the TLR3 ligand dsRNA complexed to CALV and in combination with VLPs (CALV(dsRNA) + VLPs) induced the strongest response. CALV(dsRNA) + VLPs induced the highest titers against the recombinant vaccine antigens clade B Bal gp120 and pr55 Gag. Additionally, CALV(dsRNA) + VLPs induced cross-clade antibodies, represented by high titers of antibody to clade c 96ZM651 gp120. CALV(dsRNA) + VLPs induced predominantly IgG2c over IgG1, a response associated with T helper type 1 (Th1)-like cytokines. In turn, CALV(dsRNA) + VLP immunized mice generated the most potent neutralizing antibodies against HIV strain MN.3. Finally, at time of sacrifice, a significant increase in germinal center B cells and T follicular cells was detected in mice which received CALV(dsRNA) + VLPs compared to PBS. Our results indicate that CALV(dsRNA) is a superior adjuvant for HIV VLPs in generating a Th1-like immunoglobulin profile, while prolonging lymph node germinal centers, T follicular cells and generating neutralizing antibodies to a highly sensitive tier 1A variant of HIV.

Introduction

Virus-Like Particles (VLPs) are replication-incompetent subunit vaccines, which lack a genome, but maintain the original surface antigenic composition of the virus. HIV VLPs are composed of the HIV Gag structural protein and the membrane components of the host cell, which typically includes the expression of the exogenous HIV envelope membrane component gp160, cleaved into functional gp120/gp41 [1], [2]. HIV VLPs have previously been shown to act as potent immunogens, capable of activating dendritic cells and macrophages, as well as directly activating B cells [3], [4], [5]. Recently, our lab identified intranasal prime, sub-cheek boost as a novel route of vaccine administration, which when coupled with HIV VLPs and a liposomal formulation containing the toll-like-receptor (TLR) 4 ligand monophosphoryl lipid A (MPLA), induced high serum immunoglobulin (IgG) titers and a Th1-like IgG profile [6].

TLR ligands make ideal adjuvants due to their receptor expression on antigen presenting cells (APCs), strong innate immune activation, and lack of host expression [7], [8]. Ligands for TLRs 3, 4, 5, 7/8, and 9 have independently been combined with HIV VLPs in previous studies, but no direct comparison has been undertaken [6], [9], [10], [11]. Additionally, how these adjuvants affect the adaptive immune system, in particular B cell hypermutation and class switching, over an extended period of time is glaringly understudied.

Germinal centers, located in the lymph nodes (LNs) and spleen, are the regions in which B cells undergo antigen specific somatic hypermutation and antibody class switching [12], [13]. To maintain the germinal center, germinal center B cells are supported by a subset of T cells known as T follicular (TFH) cells, which through direct cell signaling and cytokine secretion maintain B cell maturation [14], [15], [16]. B cell maturation, in particular somatic hypermutation, is critical for the development of B cells capable of secreting high affinity HIV neutralizing antibodies [17], [18].

In this study, we evaluated and compared the adjuvant properties among a panel of TLR ligands and assessed their ability to facilitate VLP immunogenicity by producing Th1-like class switching, high sera IgG titers, maintain germinal centers, and TFH cells which contribute to the antibody production against the target antigens. By targeting distinct TLRs in combination with our VLPs, we aimed to deduce how TLR cellular localization and activated downstream pathways could induce the optimal VLPs vaccine immune response.

Section snippets

Animals

Female C57BL/6 mice from Jackson Labs (Farmington, CT) were purchased and used at 8 weeks of age. All mice were maintained under specific pathogen-free conditions in the animal facilities of Baylor College of Medicine and in accordance with the animal protocol approved by Institutional Animal Care and Use Committee (IACUC).

Western blot

Western blot was performed as described previously [6]. Envelope protein was detected with primary human monoclonal antibody to V3 of HIV-1 Env (447-52D; NIH AIDS Reagent

VLPs induce robust cellular immune response

Mice were immunized with one intranasal prime followed by two sub-cheek boosts of VLPs with or without the indicated adjuvants, and then retained for an additional 12 weeks following the final boost to monitor sera immunoglobulin concentrations (Table 1; Supplementary Fig. 1B). VLPs consisted of HIVIIIB Gag and HIV-1 BaL gp160, which was cleaved into gp120 and gp41; each mouse received an average of 2.24 μg per immunization of BaL gp120 envelope (Supplementary Fig. 1A). Two weeks after the second

Discussion

The aim of this study was to investigate a panel of TLR ligands as adjuvants for our HIV VLPs, assessed by longitudinal measurements of serum antibodies and germinal center B cells. By measuring serum antibody titers for an additional 3 months after the last immunization, we were able to better determine which adjuvants could induce long-lasting Th1-like IgG class-switching, as well as observe sustained germinal center activity.

By measuring serum IgG titers over a 3 month period after

Conflict of interest

The authors have no conflicts of interest to report.

Acknowledgements

We would like to thank Dr. Paul Spearman at Emory University for the VLP producing cell lines. VA merit Grants 1I01BX001474-01A1 (PI: Yao), NIH SBIR R43AI104073 (PI: Fujii). Phoebe Lewis received IMSD support R25GM56929. Neutralization assays were funded by NIAID-NIH under contract # HHSN27201100016C (to DCM). This project was supported by the Cytometry and Cell Sorting Core at Baylor College of Medicine with funding from the NIH (P30 AI036211, P30 CA125123, and S10 RR024574) and the expert

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