Acute antagonism in three-drug combinations for vaginal HIV prevention in humanized mice

BLT hu-mouse study design and efficacy endpoints

The BLT hu-mouse model is well-established in HIV-1 prevention research^20,24. Humanization of the immune system in these mice is achieved by implanting human fetal liver and thymus tissues under the kidney capsule of immunodeficient NOD-scid gamma chain knockout (NSG), followed by administration of autologous human fetal liver CD34⁺ cells (human hematopoietic stem and progenitor cells, HSPCs). T-cell education occurs in the human thymic tissue leading to complete systemic reconstitution of all major human hematopoietic lineages, including T, B, monocyte/macrophage, dendritic, and natural killer cells. Importantly, the extensive systemic and genital mucosal reconstitution with human lymphoid cells renders female BLT hu-mice susceptible to vaginal HIV-1 infection. We verified the degree of humanization of the BLT mice at 20 weeks of age, 10 weeks post-CD34⁺ HSPC injection, as in previous studies^25,26,27. Only mice that met the humanization targets determined previously²⁸ (> 65% of CD45⁺ cells and > 70% of CD3⁺ and CD4⁺ cells) were used in infection studies.

The overall vaginal HIV-1 prevention study design using female BLT hu-mice has been described previously^25,26,27, and is shown schematically in Fig. 1. Either 8 or 10 mice per group were employed. The statistical power resulting from these group sizes was calculated using two-sided binomial tests, and has been reported previously²⁷. Viral RNA was measured in plasma at 1, 2, 3, 6, and 12 weeks post challenge to determine if infection occurred.

Experimental design of BLT hu-mouse vaginal HIV-1 prevention studies. Vaginal application of drug solution in phosphate-buffered saline (PBS, green box) was followed by the HIV-1 exposure (yellow box) within 30 min (typically 15–25 min). Peripheral blood samples were collected at the indicated times (red box) and HIV-1 viral load measured by qPCR.

A diagonal, constant, equipotency ratio combination design²¹ (see Supplementary Data S1–S4 for drug dosing concentrations) was used to minimize the number of animal groups used, while still allowing the quantitative study endpoints to be met. Typically, 5–6 drug concentrations per group were used (N = 8–10 mice each, vide supra). Initially, we determined the ED₅₀ values (the dose required to achieve 50% protection from HIV-1 infection) for drugs administered individually, allowing combination experiments to be designed. The ED₅₀ and ED₉₀ values calculated here allow for standardized comparison of drug potencies and form important inputs to the empirical analysis of drug combination effects (vide infra). However, they should not be viewed at concentration targets for vaginal HIV-1 PrEP products, as these would need to be higher, predicated by safety, to maximize duration of efficacy.

For three-drug combinations, we created equipotency ratio combinations based on multiples (1/3, 1, and 3) of the individual drugs’ vaginal ED₅₀ values (Supplementary Data S3). A subsequent study using three additional dosing groups determined based on the pilot study results was used to fill in gaps in the dose–response relationships (Supplementary Data S4). This approach was facilitated by the high reproducibility in humanization of the mice observed in previous studies^25,26,27.

Dose–response and slope parameters for single antiretroviral drug regimens

We have previously measured the vaginal HIV-1 prevention dose–response characteristics of single agents spanning a range of antiviral mechanisms of action, namely: TDF²⁵, a prodrug and nucleotide analog reverse transcriptase inhibitor competing with adenosine monophosphate; FTC²⁵, a nucleoside analog reverse transcriptase inhibitor competing with cytidine; C5A²⁶, a broadly antiviral, 18-amino acid, linear peptide that disrupts the viral membrane; and VRC01-N²⁷, a broadly neutralizing antibody (bNAb) against HIV-1. Here, we add efficacy data for two further ARV drugs from different mechanistic classes to our in vivo HIV-1 prevention dataset.

The dose–response relationships for the chemokine co-receptor 5 (CCR5) antagonist maraviroc (MVC, N = 8 per dosing group, 5 groups, Supplementary Data S1) and the integrase strand transfer inhibitor (ISTI) elvitegravir (EVG, N = 8 per dosing group, 5 groups, Supplementary Data S2) are presented in Fig. 2.

Dose–response curves for vaginal HIV-1 challenge studies in BLT hu-mice. Plots of efficacy versus dose of (A) MVC (N = 8 per dosing group, 5 groups) and (C) EVG (N = 8 per dosing group, 5 groups) applied prior to HIV-1 challenge, analyzed using a variable slope model (four-parameter dose–response curve) that does not constrain the Hill slope. Median-effect plots for the corresponding vaginal HIV-1 efficacy studies using (B) MVC and (D) EVG dosing represent a different model for data analysis. F_a, fraction affected; F_u, fraction unaffected; D, dose (nM).

The compounds exhibited sigmoidal dose–effect HIV-1 prevention curves analogous to those observed in our previous studies with TDF, FTC, C5A, and VRC01-N. Key pharmacodynamic (efficacy) parameters from the single drug systems shown in Fig. 2, along with data from previous studies^25,26, re-analyzed using boundary conditions consistent with the current analysis (see “Methods”), were calculated using the median-effect Eq. ²¹ and are presented in Table 1.

The median-effects plots^21,22 allowed a number of key parameters to be calculated (Fig. 2B,D, Table 1), based on the condition that the mass-action principle be followed by the system under investigation. This criterion, defined by R² values greater than 0.9 for in vivo studies^21,22, was met in the above vaginal challenge studies (Table 1). The x-intercept [y = log (F_a/F_u) = 0] corresponds to the potency parameter, D_m, or the dose needed to achieve the median-effect, denoted here as ED₅₀. The slope, or m-value, is the shape parameter and defines the sigmoidicity of dose–effect curve. In cases when m = 1, the dose–effect curve is hyperbolic (e.g., Michaelis–Menten kinetics), while it is sigmoidal when m ≠ 1. The greater the m-value, the greater the sigmoidicity leading to steeper dose–response relationship (i.e., lower increase in dose concentration needed to achieve a given increase in effect, F_a), a desirable characteristic for HIV-1 PrEP. Finally, m < 1 represents a relatively rare system, typically suggestive of allosterism via drug binding at receptor or enzyme sites (i.e., negative cooperativity).

We selected EVG and C5A as antiviral agents for further evaluation as triple-drug combinations with TDF-FTC (vide infra) based on the understanding that both agents have widely disparate mechanisms of action. The ISTI EVG acts in HIV-1 target cells supporting viral replication, while the antiviral peptide C5A disrupts the viral envelope before HIV-1 binds host cells^26,29. We omitted MVC from additional studies in part based on funding limitations, and because it had ca. fivefold lower potency compared to EVG (ED₉₀, Table 1). Council et al. used the BLT hu-mouse model to show that topically applied MVC prevented vaginal HIV-1 infection in the presence of semen³⁰. Both EVG and MVC are FDA-approved ARV agents to treat HIV-1 infection that are being explored for vaginal HIV-1 prevention^18,31,32,33 and we plan to include MVC in future studies.

Empirical analysis of drug combination effects on HIV-1 preventative efficacy

The median-effect (Chou-Talalay) model based on mass action^21,22 integrates foundational biochemical and biophysical equations (see “Discussion”). It does not require a preexisting knowledge of underlying mechanisms and is used for quantitative pharmacodynamic analysis of dose–effect relationships in complex biological systems. The analytical approach is particularly powerful for empirically unraveling the effects of two or more agents administered in combination. The median-effect model was applied to analyze data from vaginal HIV-1 prevention studies in BLT hu-mice dosed topically with TDF-FTC-EVG (Fig. 3) and TDF-FTC-C5A (Fig. 4). The goal of these studies was to determine if the slight-moderate antagonism observed previously²⁵ between the nucleos

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