Determinants of successful AAV-vectored delivery of HIV-1 bNAbs in early life

Abstract

Despite advances in HIV-1 prophylaxis, vertical transmission remains a pressing problem in developing countries¹. Given the promise of broadly neutralizing antibodies (bNAbs) for HIV-1 prevention², we hypothesized that neonatal delivery of bNAbs using adeno-associated virus (AAV) could provide durable HIV-1 immunity during infancy. Here, using infant rhesus macaques (Macaca mulatta) as a model, we show that a one-time administration of an AAV vector encoding bNAb 3BNC117 at birth led to sustained bNAb expression for more than three years without redosing. This approach significantly protected both infant and pre-adolescent rhesus macaques from infection with simian-human immunodeficiency virus in mucosal challenge models that mimic HIV-1 transmission through breastfeeding and sexual intercourse. Age at the time of AAV-3BNC117 administration was a main determinant of success and was inversely correlated with the incidence of host anti-drug antibodies that restricted bNAb expression. Consistent with principles of neonatal tolerance^3,4, newborn rhesus macaques exhibited higher levels of bNAb expression than older infants and juveniles following AAV-3BNC117 dosing. Furthermore, in utero exposure to recombinant 3BNC117 suppressed anti-drug antibodies and improved AAV-vectored delivery of this bNAb in older infants. Thus, our results suggest that neonatal and fetal immunological tolerance can be leveraged to improve postnatal AAV delivery of HIV-1 bNAbs in primates. Since years-long HIV-1 immunity can be generated in rhesus macaques from a one-time AAV vector administration at birth, future studies should evaluate the ability of this strategy to prevent perinatal and adolescent HIV-1 infections in humans.

Fig. 1. Serum concentrations of AAV-expressed HIV IgG biologics in groups 1–3 and outcome of oral SHIV-AD8_EO challenges.

a–i, Serum concentrations of rh-eCD4-IgG2-LS (a–f) or rh-3BNC117-IgG1-LS (g–i) and ADA levels (a–i) in infant rhesus macaques in group 1 (rh1-1 (a), rh1-2 (b) and rh1-3 (c)), group 2 (rh2-1 (d), rh2-2 (e) and rh2-3 (f)), and group 3 (rh3-1 (g), rh3-2 (h) and rh3-3 (i)). ADA responses for groups 1–3 are depicted as the serum immunoglobulin reactivity (absorbance values at 450 nm (A_{450 nm})) against plate-bound rh-eCD4-IgG2-LS or rh-3BNC117-IgG1-LS. j, Kaplan–Meier analysis of SHIV acquisition in each experimental group (groups 1–3) versus the control group (group 4) after oral challenges with increasing doses of SHIV-AD8_EO expressed as Gag p27 content. P values were calculated using the Mantel–Cox test. k,l, log₁₀-transformed plasma viral loads in the group 4 macaques (n = 6) (k) and in the only group 1 (l) monkey (rh1-1) that acquired infection. Empty red bars in l indicate the post-infection serum rh-eCD4-IgG2-LS concentrations. Source Data

Fig. 2. Age affects the outcomes of gene therapy with an AAV-8-rh-3BNC117-IgG1-LS vector.

a–j, The AAV-8-rh-3BNC117-IgG1-LS vector was administered to rhesus macaques across four age groups: group 5 (a,b; 48 h, except for rh5-6 (see below)); group 6 (c,d; approximately 2 years); group 7 (e,f; 4 weeks); group 8 (g,h; 8 weeks); and group 9 (i,j; 12 weeks). LLOQ, lower limit of quantification. a,c,e,g,i, Serum rh-3BNC117-IgG1-LS concentrations. b,d,f,h,j, Anti-rh-3BNC117-IgG1-LS responses, or ADAs, in each group. k, Serum concentrations of rh-3BNC117-IgG1-LS at week 20 in the group 5 newborns (n = 5) and group 6 juveniles (n = 6). l, Comparison of cumulative levels of ADAs produced by the group 5 newborns and group 6 juveniles during the 20 weeks of follow-up. AUC, area under the curve. In k,l, the group 5 infant rh5-6 was not included in the comparisons because it received the AAV-8-rh-3BNC117-IgG1-LS vector 4 weeks after birth. m, Monkeys in groups 3 and 5–9 were divided into three age brackets and their serum rh-3BNC117-IgG1-LS concentrations at week 20 were compared. In k–m, bars correspond to medians and P values were two-sided and calculated using the Mann–Whitney U-test. P values in brackets were calculated without the group 6 outlier rh6-6. n, Correlation between the age (in weeks) of each monkey at the time of AAV inoculation and serum rh-3BNC117-IgG1-LS concentration at week 20. Source Data

Fig. 3. In utero exposure to recombinant forms of rh-3BNC117-IgG1 prevents ADAs and improves bNAb expression following postnatal gene therapy with AAV-8-rh-3BNC117-IgG1-LS.

While the macaques in groups 10 and 11 were in their final weeks of gestation, their mothers were treated intravenously with recombinant rh-3BNC117-IgG1-LS or rh-3BNC117-IgG1, leading to transplacental transfer of these molecules. The macaques that were prenatally exposed to the bNAb were delivered two weeks later and then treated with the AAV-8-rh-3BNC117-IgG1-LS vector at 8 weeks (group 10) or 12 weeks (group 11) of age. a,c, Serum rh-3BNC117-IgG1-LS concentrations in group 10 (a) and group 11 (c). ADAs were assessed as described in Fig. 1 for group 10 (b) and group 11 (d). All samples from groups 7–11 were screened for anti-rh-3BNC117-IgG1-LS antibodies in the same 384-well enzyme-linked immunosorbent assay (ELISA) plate. e,f, Cumulative serum rh-3BNC117-IgG1-LS concentrations (e) and ADA levels (absorbance values of anti-rh-3BNC117-IgG1-LS reactivity in serum) (f) measured over the first 20 weeks post-intervention were compared between infant macaques exposed to recombinant forms of rh-3BNC117-IgG1 in utero (groups 10 and 11) and age-matched bNAb-naive infants (groups 8 and 9). Bars correspond to medians. All P values were two-sided and calculated using the Mann–Whitney U-test. Source Data

Fig. 4. Rhesus macaques treated with AAV-8-rh-3BNC117-IgG1-LS at birth develop and maintain levels of bNAb expression that can block rectal SHIV acquisition in pre-adolescence.

Four out of the seven AAV-treated macaques in groups 1–3 that resisted oral challenges with SHIV-AD8_EO were kept alive for up to four years, and AAV-driven transgene expression in serum was monitored. These macaques were not re-dosed with AAV vectors after the neonatal period. a–d, Serum concentrations of rh-3BNC117-IgG1-LS (a,b) or rh-eCD4-IgG2-LS (c,d) and NAb titres against SHIV-AD8_EO for rh3-1 (a), rh3-2 (b), rh1-2 (c), and rh2-3 (d). Date of birth (DOB) and date of the last rh-3BNC117-IgG1-LS or rh-eCD4-IgG2-LS measurement are shown. ID₅₀, half-maximal infectious dose. e–i, The six macaques in group 5 were kept alive beyond the 20-week follow-up period described in Fig. 2 and their serum concentrations of rh-3BNC117-IgG1-LS (e) and NAb titres against SHIV-AD8_EO (f) were monitored until the animals reached approximately 2.5 years of age. Beginning at weeks 133–140 post-intervention, the group 5 monkeys and six controls (group 12) were subjected to repeated intrarectal (IR) challenges with a fixed marginal dose of SHIV-AD8_EO. g, Kaplan–Meier analysis of SHIV acquisition in groups 5 and 12. The P value was calculated using the Mantel–Cox test. h,i, Plasma viral loads in the group 12 macaques (n = 6) (h) and in the only group 5 monkey (rh5-2) (i) that acquired infection. Source Data

Extended Data Fig. 1. Layout of AAV vector inoculations and timing of oral SHIV challenges in Groups 1–4.

a, The three infants in Group 1 were treated intramuscularly (IM) at birth with the indicated dose of an AAV-1 vector expressing rh-eCD4-IgG2-LS. b, The three infants in Group 2 were treated intravenously (IV) at birth with the indicated dose of an AAV-8 vector expressing rh-eCD4-IgG2-LS. Four weeks later, the Group 2 macaques were injected IM with the same dose of the same AAV-1/rh-eCD4-IgG2-LS vector administered to the Group 1 animals. c, The three infants in Group 3 were injected IM at birth with the indicated dose of an AAV-8 vector expressing rh-3BNC117-IgG1-LS. As the nine infants in Groups 1–3 approached 20 weeks of age, a per-protocol transition to paired housing necessitated the removal of one animal from the study. Because serum concentrations of rh-3BNC117-IgG1-LS had fallen below detection limits after week 12 in the Group 3 infant rh3-3, this animal was removed from the study. d, The six AAV vector-naïve infants in Group 4 were matched in age to those in Groups 1–3. Beginning at postnatal weeks 30–34, all the AAV-treated RMs in Groups 1–3 (except for rh3-3) and the Group 4 controls were subjected to weekly oral challenges with escalating doses of SHIV-AD8_EO.

Extended Data Fig. 2. Kinetics of weight gain through the first year of life in macaques in Groups 1–3.

Weight (kg) increased at an expected rate in the AAV vector-treated infant macaques in Group 1 (a), Group 2 (b), and Group 3 (c) during the first year of life. The timing of each AAV vector inoculation is indicated by vertical dotted lines and color-coded hexagons. Source Data

Extended Data Fig. 3. Serological neutralizing activity against SHIV-AD8_EO in macaques in Groups 1–3.

Sera from the macaques in Group 1 (a-c), Group 2 (d-f), and Group 3 (g,h) were tested for their ability to neutralize the in vitro infectivity of SHIV-AD8_EO in TZM-bl cells. Neutralizing titers [i.e., inhibitory dilutions at which 50% of neutralization (ID₅₀) was observed] were derived from these assays and plotted against the right y-axis of each panel. As a reference, the serum concentrations of rh-eCD4-IgG2-LS (a-f) or rh-3BNC117-IgG1-LS (g,h) were plotted against the left y-axis. Sera from the Group 3 macaque rh3-3 were not assayed for anti-SHIV-AD8_EO neutralizing antibodies because this animal was euthanized before the start of the oral SHIV challenge phase (gray shaded boxes). Source Data

Extended Data Fig. 4. Kinetics of AAV-8 seroconversion in Groups 5 and 6.

Total IgG reactivity to the AAV-8 capsid was measured by ELISA using a fixed dilution (1:1,000) of serum collected at baseline (day 0) and every two weeks thereafter until the last follow up at week 20. All samples from the Group 5 (n = 6; a) and Group 6 (n = 6; b) animals were tested in the same 384-well ELISA plate and the absorbance values measured at 450 nm for each sample were plotted against time. c, Geometric means of absorbance values measured for Group 5 (yellow hexagons) and Group 6 (gray hexagons) were plotted against time. The two-sided P value was calculated using the Mann-Whitney U test and resulted from comparing Groups 5 and 6 in terms of their cumulative levels of anti-AAV-8 IgG reactivity (area under curve analysis of absorbance values) measured over the 20 weeks of follow-up. Source Data

Extended Data Fig. 5. Layout and timeline of experiments for the pregnant females in Groups A–C and their offspring in Groups 7–11.

Sixteen pregnant female rhesus macaques at similar gestational stages were selected on the basis of having little or no serum reactivity to AAV-8 by ELISA. These animals were then divided into three groups depending on whether they were treated with recombinant forms of rh-3BNC117-IgG1. The Group A dams (n = 8) received no bNAb infusion and underwent cesarean (c)-section when their pregnancies reached term at gestational week 24. The only exceptions were dams rhA-1 and rhA-3, which delivered their babies vaginally ahead of schedule. The dams in Groups B and C (n = 4 each) were treated intravenously with 30 mg/kg of recombinant rh-3BNC117-IgG1-LS or rh-3BNC117-IgG1, respectively, at gestational week 22 and then underwent c-section two weeks later. The offspring of the dams in Groups A-C were transferred to the nursery after birth and then assigned to Groups 7–11. The infants in Groups 7–11 were treated intramuscularly (IM) with 2.0 × 10¹² genome copies ml^–1 of an AAV-8/rh-3BNC117-IgG1-LS vector at postnatal weeks 4, 8, or 12, as depicted.

Extended Data Fig. 6. Univariate differential expression analysis of whole blood transcripts in Groups 5 and 6.

a-d, Volcano plots depicting the statistical significance (y-axes; negative log₁₀-transformed P values) and fold change (FC) in gene expression (x-axes; log₂-transformed FC) in differentially expressed genes (DEGs) measured in Group 5 (a,c) or Group 6 (b,d) on day 3 vs baseline (day 0) (a,b) or at week 4 vs baseline (c,d). Each data point represents one DEG. Those colored in red have both an adjusted P value < 0.1 and a log₂ FC ± 0.5. DEGs colored in blue and green meet either the P value or the log₂ FC criterion, respectively, but not both. Gray DEGs meet neither criterion and, therefore, are not significant (NS). The Wald test default method in Sleuth was used to assess whether the estimated coefficients (i.e., FCs) for each DEG was significantly different from zero. All P values are two-sided e,f, Heatmaps depicting differences in expression of top significant differentially expressed genes in Group 5 (e) and Group 6 (f) in week 4 vs baseline (day of AAV inoculation) comparisons. The color gradient from blue to red is obtained using z-transformed transcript per million values, where negative (blue) and positive (red) identify low and highly expressed genes, respectively. g,h, Dot plots showing overrepresented pathways by genes that are upregulated at week 4 in Group 5 (g) and Group 6 (h), as annotated in the Reactome database.

Extended Data Fig. 7. Univariate differential expression analysis of whole blood transcripts in Groups 7–9.

For this analysis, Groups 8 and 9 were pooled, whereas Group 7 was examined separately. a,b, Volcano plots, as described in Extended Data Fig. 6, depicting the statistical significance and fold change (FC) in gene expression in differentially expressed genes measured at baseline (day 0) in Group 7 (a) and Groups 8 + 9 (b). The Wald test default method in Sleuth was used to assess whether the estimated coefficients (i.e., FCs) for each DEG was significantly different from zero. All P values are two-sided. NS, not significant. c,d, Dot plots showing overrepresented pathways by genes that are upregulated at week 4 in Group 7 (c) and Groups 8 + 9 (d), as annotated in the Reactome database.

Extended Data Fig. 8. Pharmacokinetics of recombinant forms of rh-3BNC117-IgG1 administered to the pregnant dams in Groups B and C.

a-h, Serum concentrations of rh-3BNC117-IgG1-LS or rh-3BNC117-IgG1 in the Group B (a-d) and Group C (e-h) dams following passive infusion of 30 mg/kg these molecules at gestational week 22 (vertical dotted lines). Two weeks later, pregnancies reached term and the animals underwent cesarean (c)-section (vertical solid line). The last two weeks of pregnancy are indicated by a gray box. i, Table listing the concentrations of rh-3BNC117-IgG1-LS or rh-3BNC117-IgG1 in maternal, cord blood, or newborn serum at the time of c-section. Source Data