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. 2019 Sep 27;9(10):1389.
doi: 10.3390/nano9101389.

Calcium Phosphate Nanoparticle-Based Vaccines as a Platform for Improvement of HIV-1 Env Antibody Responses by Intrastructural Help

Dominik Damm  1 , Leonardo Rojas-Sánchez  2 , Hannah Theobald  3 , Viktoriya Sokolova  4 , Richard T Wyatt  5 , Klaus Überla  6 , Matthias Epple  7 , Vladimir Temchura  8
Affiliations

Calcium Phosphate Nanoparticle-Based Vaccines as a Platform for Improvement of HIV-1 Env Antibody Responses by Intrastructural Help

Dominik Damm et al. Nanomaterials (Basel). .

Abstract

Incorporation of immunodominant T-helper epitopes of licensed vaccines into virus-like particles (VLP) allows to harness T-helper cells induced by the licensed vaccines to provide intrastructural help (ISH) for B-cell responses against the surface proteins of the VLPs. To explore whether ISH could also improve antibody responses to calcium phosphate (CaP) nanoparticle vaccines we loaded the nanoparticle core with a universal T-helper epitope of Tetanus toxoid (p30) and functionalized the surface of CaP nanoparticles with stabilized trimers of the HIV-1 envelope (Env) resulting in Env-CaP-p30 nanoparticles. In contrast to soluble Env trimers, Env containing CaP nanoparticles induced activation of naïve Env-specific B-cells in vitro. Mice previously vaccinated against Tetanus raised stronger humoral immune responses against Env after immunization with Env-CaP-p30 than mice not vaccinated against Tetanus. The enhancing effect of ISH on anti-Env antibody levels was not attended with increased Env-specific IFN-γ CD4 T-cell responses that otherwise may potentially influence the susceptibility to HIV-1 infection. Thus, CaP nanoparticles functionalized with stabilized HIV-1 Env trimers and heterologous T-helper epitopes are able to recruit heterologous T-helper cells induced by a licensed vaccine and improve anti-Env antibody responses by intrastructural help.

Keywords: B-cell targeting; HIV-1 Env trimers; intrastructural help; nano-vaccines.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Production and characterization of soluble HIV-1 Env constructs and Env-p30-VLPs. (A) Overview of different membrane-embedded and soluble Env constructs. The native BG505 gp160 (left), that is composed of three gp120 (grey) – gp41 (blue) heterodimers, has been used previously to create a soluble stabilized recombinant trimer (BG505 NFL2P gp140; middle left) by truncation at amino acid 664 and introduction of both a point mutation (I559P) indicated with an asterisk (*) and a flexible linker (2xG4S). As a purification control, we produced a BG505 NFL2P gp120 monomer (middle right) by truncation of the protein downstream of the flexible linker. A recombinant VSV-G transmembrane and cytoplasmic domain (TM/CD; red) was introduced downstream of the gp41 ectodomain to create a membrane-bound form of the stabilized trimer for production of Env-VLP-p30 particles (BG505 NFL2P gp140-GTMCD; right). (B) Native PAGE of purified gp140 and gp120. Three gp140 molecules formed a globular trimer molecule represented by a major band at 700 kDa. The gp120 subunit was expressed as monomeric or dimeric proteins, but did not form trimers. (C,D) Schematic overview of the plasmids used for Env-VLP-p30 production. (C) BG505 NFL2P gp140-GTMCD construct. Colors are matched to their respective protein domains in Figure 1A. (D) Hgpsyn-TTp30 construct: the nucleotide sequence for the p30 peptide was introduced between the HIV-1 p17 matrix protein and the spacer protein 1 followed by the capsid protein p24. (E) Reducing SDS-PAGE and Western Blot of purified soluble Env proteins and Env-VLP-p30.
Figure 2
Figure 2
Scanning electron micrographs and dynamic light scattering (DLS) particle size distribution of the prepared nanoparticles: (A) Env-CaP, (B) Env-CaP-p30, (C) Env-CaP-CpG.
Figure 3
Figure 3
Activation of naïve B-cells in vitro. Naïve B-cells were isolated from PGT121 (A) or wt (B) mice and stimulated with Env trimers (8, 40 or 200 ng of Env/mL), Env-CaP nanoparticles (8, 40 or 200 ng of Env/mL), Env-VLPs (200 ng of Env/mL), or LPS (2 µg/mL). After 18h incubation, the cells were stained with a viability dye and with anti-CD19 and anti-CD69 antibodies. The histograms represent the expression of CD69 on the surface of viable CD19-positive B-cells. The numbers on the histograms indicate the percentage of the gated CD69-positive cells and medians of the total CD69 fluorescence intensity.
Figure 4
Figure 4
Improvement of Env-specific IgG subtype responses after Env-CaP-p30 immunizations by intrastructural help from Tetanus toxoid-specific T-helper cells. (A,B) IgG1 (A) and IgG2c (B) Env-specific antibody responses were measured in sera from wt mice primed twice with DPBS or Tetanol (ISH) and boosted 3 times with different CaP nanoparticles or Env-VLP-p30. (C,D) Percentages of CD4 T-cells producing IFN-γ (C) and TNF-α (D) from differently immunized wt animals after in vitro re-stimulation with p30 peptide were measured by intracellular cytokine staining. (E) p30-specific IL-5 cytokine secretion as determined by ELISA. The columns represent the mean values of six animals ± SEM. * p < 0.05; ** p < 0.001; *** p < 0.0005; **** p < 0.0001; one-way ANOVA with Tukey multiple comparison post-hoc test.
Figure 5
Figure 5
Characterization of anti-Env immune responses after heterologous ISH and CpG adjuvantation. (A) Env-specific antibody responses were measured in sera from wt mice primed twice with Tetanol and boosted 3 times with either Env-CaP-p30 or Env-CaP-CpG. The columns represent the mean values of six animals ± SEM. **** p < 0.0001; one-way ANOVA with Tukey multiple comparison post-hoc test. (B) Env-specific IgG1/IgG2c ratios of individual mice. The columns represent the mean values of six animals ± SEM. **** p < 0.0001; unpaired Student t-test. (C–E) Percentages of CD4 T-cells producing IFN-γ (C), TNF-α (D) and IL-2 (E) from differently immunized wt animals after in vitro re-stimulation with Env-VLPs were measured by intracellular cytokine staining. The columns represent the mean values of six animals ± SEM. **** p < 0.0001; one-way ANOVA with Tukey multiple comparison post-hoc test.
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References

    1. Temchura V., Tenbusch M. The two faces of vaccine-induced immune response: Protection or increased risk of HIV infection?! Virol. Sin. 2014;29:7–9. doi: 10.1007/s12250-014-3419-9. - DOI - PMC - PubMed
    1. Chung A.W., Ghebremichael M., Robinson H., Brown E., Choi I., Lane S., Dugast A.S., Schoen M.K., Rolland M., Suscovich T.J., et al. Polyfunctional Fc-Effector Profiles Mediated by IgG Subclass Selection Distinguish RV144 and VAX003 Vaccines. Sci. Transl. Med. 2014;6:228ra38. doi: 10.1126/scitranslmed.3007736. - DOI - PubMed
    1. Kim J.H., Excler J.-L., Michael N.L. Lessons from the RV144 Thai Phase III HIV-1 Vaccine Trial and the Search for Correlates of Protection. Annu. Rev. Med. 2015;66:423–437. doi: 10.1146/annurev-med-052912-123749. - DOI - PubMed
    1. Excler J.-L., Ake J., Robb M.L., Kim J.H., Plotkin S.A. Nonneutralizing Functional Antibodies: A New “Old” Paradigm for HIV Vaccines. Clin. Vaccine Immunol. 2014;21:1023–1036. doi: 10.1128/CVI.00230-14. - DOI - PMC - PubMed
    1. Ackerman M.E., Mikhailova A., Brown E.P., Dowell K.G., Walker B.D., Bailey-Kellogg C., Suscovich T.J., Alter G. Polyfunctional HIV-Specific Antibody Responses Are Associated with Spontaneous HIV Control. PLoS Pathog. 2016;12:e1005315. doi: 10.1371/journal.ppat.1005315. - DOI - PMC - PubMed