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. 2023 Aug 16:18:4635-4645.
doi: 10.2147/IJN.S412915. eCollection 2023.

Combined Nano-Vector Mediated-Transfer to Suppress HIV-1 Infection with Targeted Antibodies in-vitro

Xin Yao  1 , Qingyu Wang  1 , Changge Han  1 , Jiaojiao Nie  1 , Yaotian Chang  1 , Lipeng Xu  1 , Bingya Wu  1 , Jingtian Yan  1 , Zhiyuan Chen  1 , Wei Kong  1   2 , Yuhua Shi  1 , Yaming Shan  1   2
Affiliations

Combined Nano-Vector Mediated-Transfer to Suppress HIV-1 Infection with Targeted Antibodies in-vitro

Xin Yao et al. Int J Nanomedicine. .

Abstract

Introduction: Broadly neutralizing antibodies (bNAbs) have the ability to neutralize a considerable breadth of genetically diverse human immunodeficiency virus (HIV) strains. Passive immunization can potentially provide protection against HIV infection in animal models. However, the direct antibody infusion effect is limited due to the short half-life and deficient immunogenicity of the antibody. As an alternative strategy, we propose the use of nano viral vectors, specifically the adeno-associated virus (AAV), to continuously and systematically produce bNAbs against HIV.

Methods: Plasmids expressing bNAbs PG9, PG16, 10E8, and NIH45-46 antibodies were constructed, targeting three different epitopes of HIV. Additionally, the bNAbs gene mediated by rAAV8 was administered to generate long-term expression with a single injection. We established both single and combined immunization groups. The neutralizing activity of antibodies expressed in mice sera was subsequently evaluated.

Results: The expression of bNAbs in BALB/c mice can last for >24 weeks after a single intramuscular injection of rAAV8. Further studies show that neutralization of the HIV pseudovirus by sera from co-immunized mice with rAAV8 expressing 10E8 and PG16 was enhanced compared with mice immunized with 10E8 or PG16 alone.

Conclusion: The prolonged expression of neutralizing antibodies can be maintained over long periods in BALB/c mice. This combined immunization is a promising candidate strategy for HIV treatment.

Keywords: AAV delivery; HIV therapy; combined immunotherapy; passive immunotherapy.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

None
Graphical abstract
Figure 1
Figure 1
Antibody expression cassettes. (A) Schematic representation of bNAb constructs. Kpn I and Bgl II restriction sites are labeled atop the schematic. The expression vector components are labeled as follows: coding regions are located between the ITRs; CAG, promoters of the encoding protein; ss, human IL2 signal sequence leading to antibody secretion; HC, heavy chain antibody gene; LC, light chain antibody gene; Furin, furin cleavage site; 2A, FMDV 2A sequence. (B) Schematic representation of AAV expression plasmid.
Figure 2
Figure 2
Characterization of purified rAAV8s. (A) Analysis of capsid proteins by SDS-PAGE indicating molecular weights of VP1 (∼87 kDa), VP2 (∼72 kDa), and VP3 (∼62 kDa). (B) The white arrows indicate the rAAV8 nano-vectors. Morphological analysis via TEM. Scale bar,100 nm.
Figure 3
Figure 3
AAV-mediated antibody expression in HEK293T cells. RFP-rAAV8 was used as a negative control. The infection of 293T cells with RFP-rAAV8 was observed under a fluorescence microscope at 0h (A and D), 48h (B and E), and 72h (C and F). Scale, 50 μm. (G) Under non-reducing conditions, full-length dimerized antibodies and (H) under reducing conditions, the heavy chain of antibodies mediated by PG9-rAAV8, PG16-rAAV8, 10E8-rAAV8, and NIH45-46-rAAV8. (I) ID50 of expressed antibodies in HEK293T cell culture medium against tier 1 pseudoviruses.
Figure 4
Figure 4
Gene expression mediated by rAAV8s in BALB/c mice. (A) Immunization schedule with a single injection of rAAV8 in BALB/c mice. (B) Biophotonic imaging of rAAV8 expressing RFP in BALB/c mouse muscle at 0, 4, 8, and 24 weeks. PBS is the corresponding control group. (C) The BG505 peptide was used to detect the concentration of PG9, PG16, and NIH45-46 antibodies. (D) Concentrations of the 10E8 antibody were assessed using gp41 protein.
Figure 5
Figure 5
Evaluation of neutralizing activity of antibodies expressed by rAAV8s in BALB/c mice. Pre-immune sera and RFP-rAAV8 were used as negative controls. ID50 of expressed antibodies in BALB/c mice sera against tier 1 and tier 2 HIV-1 isolates.
Figure 6
Figure 6
Antibody expression and neutralizing activity of antibodies expressed by combined rAAV8s. (A) The BG505 protein was used to detect the titer of co-expressed antibody. (B) The titer of the co-expressed antibody was assessed using the gp41 peptide. (C) ID50 of expressed antibodies in BALB/c mice sera against tier 1 and tier 2 HIV-1 isolates. *P < 0.05, **P < 0.01, ***P < 0.001.
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