Aerosol Delivery of Synthetic mRNA to Vaginal Mucosa Leads to Durable Expression of Broadly Neutralizing Antibodies against HIV

Abstract

There is a clear need for low-cost, self-applied, long-lasting approaches to prevent human immunodeficiency virus (HIV) infection in both men and women, even with the advent of pre-exposure prophylaxis (PrEP). Broadly neutralizing antibodies represent an option to improve HIV prophylaxis, but intravenous delivery, cold-chain stability requirements, low cervicovaginal concentrations, and cost may preclude their use. Here, we present an approach to express the anti-GP120 broadly neutralizing antibody PGT121 in the primary site of inoculation, the female reproductive tract, using synthetic mRNA. Expression is achieved through aerosol delivery of unformulated mRNA in water. We demonstrated high levels of antibody expression for over 28 days with a single mRNA administration in the reproductive tract of sheep. In rhesus macaques, neutralizing antibody titers in secretions developed within 4 h and simian-HIV (SHIV) infection of ex vivo explants was prevented. Persistence of PGT121 in vaginal secretions and epithelium was achieved through the incorporation of a glycosylphosphatidylinositol (GPI) anchor into the heavy chain of the antibody. Overall, we present a new paradigm to deliver neutralizing antibodies to the female reproductive tract for the prevention of HIV infections.

Keywords: HIV; PET/CT; SHIV; aerosol delivery; animal models; intravaginal delivery; mRNA therapeutics; synthetic mRNA.

Graphical abstract

Figure 1

Aerosolized mRNA in Water Transfects Vero Cells and Ovine Cervical Epithelium, Likely through Direct Cytosolic Entry (A) Vero cells were transfected via aerosol with H₂O (control), GFP-encoding mRNA, or GFP-encoding plasmid complexed with Lipofectamine 2000 (pDNA⁺L2k). Cells were fixed and imaged for GFP (green) at 24 h. Scale bars represent 30 μm. (B) Quantification of the GFP MFI on a per-cell basis, for each transfection condition. (C) Dye-labeled mRNA (green) was delivered via aerosol to Vero cells and fixed at 30 s, 5 min, 1 h, 2 h, and 6 h post-transfection. Cells were then stained with a pan-endosomal marker cocktail including: CD63, clathrin, caveolin, EEA1, and LAMP-1 (red). Scale bars represent 10 μm. (D) The extent of spatial overlap between the endosomal markers and mRNA at each time point, up to 1 h. The Mander’s overlap coefficient reflects the proportion of mRNA that overlaps with the endosomal markers. (E) The proportion of mRNA overlapping with the endosomal markers from 1 h to 6 h; contrast enhanced on a different scale than the acute time points in (D). (F) In live sheep, 250 μg of firefly luciferase mRNA in H₂O was delivered to the cervix as a single dose with either a high-pressure syringe or via aerosolizer. A speculum was used to visualize the cervix in sedated sheep during transfection. 24 h post-transfection, the animals were euthanized and subjected to necropsy. Luciferin was added to isolated cervix and luminescence quantified as the average radiance (p/s/cm²/sr) per animal via an IVIS Lumina imaging system. Statistical comparison was performed using two tailed Mann-Whitney non-parametric analyses. For all panels, *p < 0.05, ****p < 0.00005; error bars represent ± 95% confidence interval (CI).

Figure 2

Transfected Cells Displaying Membrane-Anchored PGT121 on Their Surface Bind SHIV Virions (A) Schematic of the PGT121-NanoLuc fusion protein, in secreted and anchored forms. NanoLuc was fused to the 3′ end of the kappa light chains (2 per antibody molecule). In the case of the membrane-anchored PGT121, a GPI anchor was fused to the 3′ end of the HC. Total MW of anchored PGT121-NanoLuc was 195 kDa (B) HEK293 cells were transfected with 1 μg of PGT121 mRNA, delivered at a 4:1 ratio of HC to LC with Lipofectamine 2000 (top row). Respiratory syncytial virus neutralizing anchored Palivizumab (aPali) antibody mRNA was used as a control comparison (bottom row). 24 h later, DyLight 680-labeled SHIV particles (red) were incubated with the transfected cells for 4 h without agitation. After washing 3×, the cells were fixed and immunostained with an anti-human secondary antibody (green). (C) Average percentage overlap between SHIV virus and anchored antibodies from panel (B), using 30 cells per condition. The error bars represent the mean ± 95% CI. (D) Anchored and secreted PGT121 was produced in Vero cells and purified. The mRNA-expressed antibodies were compared against parental PGT121 for SHIV neutralization with either a clade B or clade C SHIV isolate. Error bars represent SD. (E) Anchored and secreted PGT121 fused to NanoLuc was produced in Vero cells and purified. The mRNA-expressed NanoLuc fusion antibodies were compared against parental PGT121 for SHIV neutralization with either a clade B or clade C SHIV isolate. Error bars represent SD. (F) mRNA-expressed anchored or secreted PGT121 either with or without NanoLuc was purified, diluted, and tested for ADCC against SHIV162p3 (clade B). Complete dilution series (left) and percent maximal ADCC (right) are shown. Error bars represent SD. Percent maximum ADCC compared by Kruskal-Wallis. (G) The same antibodies from (F) were purified, diluted, and tested for ADCC against SHIV2871Nip (clade C). Complete dilution series (left) and percent maximal ADCC (right) are shown. Error bars represent SD. Percent maximum ADCC compared by Kruskal-Wallis.

Figure 3

The Entire Sheep Cervicovaginal Epithelium Is Receptive to Transfection by Large Doses of aPGT121 mRNA (A) A single 250 μg or 750 μg dose of aPGT121-NanoLuc was delivered by aerosol to the cervix. After 24 h, sheep were euthanized, FRT excised, luciferin added, and luminescence measured via IVIS. Representative images demonstrating the intensity in each animal are displayed. (B) The FRT from an untreated sheep processed in the same manner as in (A). (C) The average radiance at the cervix over the average radiance in the control animal, for each mRNA dosing group. Error bars represent the ± 95% CI. (D) In 2 animals, aerosolized dye-labeled mRNA was delivered as previously described. After 24 h, the cervix was excised and processed for immunofluorescence tissue imaging. The bottom panel represents secondary only control. DAPI, cell nuclei; white, aPGT121 mRNA; green, anti-NanoLuc antibody. Section scale bars represent 10 μm. (E) aPGT121-NanoLuc mRNA was sprayed in four consecutive 750 μg doses, beginning at the cervix (marked as “1”) and retracting caudally to distal vagina (“4”).

Figure 4

aPGT121 Persists in Vaginal Secretions out to 28 Days Post-transfection Three sheep were transfected with two doses of 750 μg of mRNA encoding for aPGT121, while another two sheep were transfected with an equal mass of sPGT121 mRNA. Vaginal secretions were collected at 1, 7, 14, 21, and 28 days post-transfection. (A) Longitudinal sampling of PGT121 concentrations over time, for all animals. The horizontal asymptote represents the limit of detection. Individual and mean values can be found in Table S1. (B) Antibody concentrations in vaginal secretions were quantified via western blot analysis, using a standard curve of purified PGT121-NanoLuc protein. The expected size of aPGT121-NanoLuc was confirmed to be around 195 kDa. For secreted antibody, the LC uncoupled from the HC, resulting in a 54 kDa band.

Figure 5

aPGT121 Persists in the FRT Mucosa at 28 Days Post-transfection (A) IVIS imaging of the excised lower FRT at 14 days and 28 days post-transfection. 28-day samples for sPGT121 transfected animals were not collected due to the low signal observed at 14 days. (B) Average radiance of the secreted and anchored PGT121-NanoLuc constructs. Mann-Whitney non-parametric analysis was used to compare the two groups (C) After 28 days, tissue samples were excised under the guidance of IVIS signal, snap-frozen, and pulverized for downstream western blot analysis. (D) Day 28 post-transfection aPGT121 concentrations in excised cervix, vagina, uterus, and caudal vagina were estimated using quantitative western blot. Five animals, euthanized at 28 days, were used in total. (E) Western blot demonstrating the characteristic aPGT121 band at 195 kDa for all regions of the FRT of one treated sheep, compared to control cervix.

Figure 6

Longitudinal PET/CT Monitoring of ⁶⁴Cu Radiolabeled aPGT121 mRNA after Aerosolized FRT Delivery (A–C) Two 125 μg doses of ⁶⁴Cu radiolabeled aPGT121 mRNA were delivered via aerosol to first the cervix, then ~3–4 cm caudally in the vagina. A total of 200 μCi of ⁶⁴Cu was administered. PET/CT imaging over 3 days was used to monitor mRNA biodistribution. (A) Representative PET/CT images of the abdomen and pelvis from 70 min to 72 h. Contrast enhancement was adjusted to reflect the high SUV signals within the FRT. (B) The total SUV in the FRT over time. (C) The ratio of the total SUV in the FRT to the total SUV contained within the entire body. (D) Whole-body PET/CT images of macaque RVg13 at 70 min and 24 h post-transfection. Numbers in white represent the total SUV within the nearby organ. Contrast enhancement was adjusted to allow visualization of draining lymph nodes.

Figure 7

aPGT121 mRNA Protects Rhesus Macaque Biopsy Explants from SHIV162p3 Challenge and Is Neutralizing in Genital Secretions (A) 250 (n = 2), 400 (n = 1), or 1,000 μg (n = 1) of aerosolized aPGT121 mRNA was delivered to the ectocervix and rostral vagina of rhesus macaques. From the 250 μg group, biopsy explants were collected from the endocervix and vagina at 24 h post-transfection, while from the 400 and 1,000 μg groups, two biopsies were collected from each of the indicated regions at 24 h post transfection. Dotted vertical line separates the two animals in the 250 μg group. Biopsies were then challenged with SHIV162p3 virus. Baseline biopsy challenge data can be found in Figure S5. (B) The neutralization activity of genital secretions at 4 h, 24 h, 48 h, 72 h, and 1-week post-transfection against clade B and C SHIV strains was evaluated using the in vitro TZM-bl assay. All macaques were treated with 250 μg of aPGT121 mRNA. Macaque RCo13 was only transfected with aPGT121 HC (i.e., no LC). The dashed horizontal line represents the lower limit of detection. Complete dilution series neutralization data can be found in Figure S6.