Characterization of Simian Immunodeficiency Virus Variants Anatomically Compartmentalized in Plasma and Milk in Chronically Infected African Green Monkeys

Abstract

Unlike human immunodeficiency virus type 1 (HIV-1)-infected humans, African-origin, natural simian immunodeficiency virus (SIV) hosts, such as African green monkeys (AGMs), sustain nonpathogenic SIV infections and rarely vertically transmit SIV to their infants. Interestingly, chronically SIV-infected AGMs have anatomically compartmentalized SIV variants in plasma and milk, whereas humans and SIV-infected rhesus monkeys (RMs), Asian-origin nonnatural SIV hosts, do not exhibit this compartmentalization. Thus, it is possible that AGM SIV populations in milk have unique phenotypic features that contribute to the low postnatal transmission rates observed in this natural host species. In this study, we explored this possibility by characterizing the infectivity, tropism, and neutralization susceptibility of plasma and milk SIVsab env variants isolated from chronically SIVsab92018ivTF-infected AGMs. AGM plasma and milk SIVsab env pseudovirus variants exhibited similar infectivities, neutralization susceptibilities to autologous and heterologous plasma, and chemokine coreceptor usages for cell entry, suggesting similar abilities to initiate infection in a new host. We also assessed the cytokine milieu in SIV-infected AGM milk and compared it to that of SIV-infected RMs. MIP-1β, granulocyte colony-stimulating factor (G-CSF), interleukin-12/23 (IL-12/23), and IL-13 trended significantly higher in SIV-infected AGM milk than in that of RMs, while IL-18 and IL-6 trended significantly higher in SIV-infected RM milk than in that of AGMs. Taken together, our findings imply that nonviral maternal factors, such as the cytokine milieu, rather than unique characteristics of SIV populations in the milk contribute to the low postnatal transmission rates observed in AGMs.

Importance: Due to the ongoing global incidence of pediatric HIV-1 infections, including many that occur via breastfeeding, development of effective vaccine strategies capable of preventing vertical HIV transmission through breastfeeding remains an important goal. Unlike HIV-1-infected humans, African green monkeys (AGMs), the natural SIV host species, sustain nonpathogenic SIV infections, rarely transmit the virus postnatally to their infants, and exhibit anatomically compartmentalized SIV populations in milk and plasma. Identifying unique features of the anatomically compartmentalized milk SIV populations could enhance our understanding of how AGMs may have evolved to avoid transmission through breastfeeding. While this study identified limited phenotypic distinctions between AGM plasma and milk SIV populations, potential differences in milk cytokine profiles of natural and nonnatural SIV hosts were observed. These findings imply the potential importance of nonviral factors in natural SIV host species, such as innate SIV/HIV immune factors in milk, as a means of naturally preventing vertical transmission.

FIG 1

Phylogenetic tree of AGM plasma and milk SIVsab env variants. Maximum-likelihood trees of all (A) and the 23 cloned and phenotypically characterized (B) SIV env sequences isolated from 4 AGMs at 5 months postinfection by single-genome amplification from plasma (PL) (red) and milk (BM) (blue) are shown. The challenge strain is shown with an open circle. Bootstrap scores indicated for branches with more than 60% reproducibility after 1,000 analyses. The scale bar represents 0.0005 nuclear substitution per site. *, SIV variants selected for cloning and phenotypic characterization.

FIG 2

Similar infectivity of plasma and milk SIVsab variants. (A) No difference between plasma and milk SIVsab variant infectivity normalized to total Gag p24 (TCID₅₀/[Gag p24]) in TZM-bl cells (Wilcoxon test; P = 0.10). (B) Strong correlation of plasma and milk neutralization responses against SIVsab92018ivTF in TZM-bl and M7-luc cells (Spearman's rank test; r = 0.95, P < 0.0001). (C) Similar infectivity of SIVsab92018ivTF in TZM-bl and M7-luc cell lines (TZM-bl, TCID₅₀/ml = 69,877; M7-luc, TCID₅₀/ml = 72,407).

FIG 3

Autologous and heterologous neutralization sensitivities of plasma and milk SIVsab variants in TZM-bl cells. (A) Neutralization of SIVsab plasma and milk variants isolated at 5 months postinfection by autologous plasma collected throughout chronic SIVsab infection. The magnitudes of autologous plasma neutralization potency were similar against milk and blood variants (dichotomized mixed effects model; P = 0.21, OR = 0.28 [0.04 to 2.12]). Red lines indicate plasma variants, and blue lines indicate milk variants. Shapes indicate the monkey from which the variants were isolated. (B) Ten of 13 plasma and 6 of 9 milk SIVsab variants were sensitive to autologous neutralization at 1 or 2 years postinfection (Fisher's exact test; P = 0.66). In general, milk and plasma variants were sensitive to the same heterologous plasma samples.

FIG 4

Susceptibility of plasma and milk SIVsab variants to CCR5 and CXCR4 chemokine receptor antagonists. (A and B) Percent neutralization by TAK (CCR5 antagonist at 10 μM) (A) and AMD (CXCR4 antagonist at 1.3 μM) (B). Replication of all variants was inhibited by TAK (CCR5 antagonist), whereas replication of 4/13 plasma variants and 0/10 milk variants was inhibited by AMD (CXCR4 antagonist) (Fisher's exact test; P = 0.10). The cutoff for detectable neutralization by TAK and AMD (dashed line) was defined as 3 standard deviations above the mean percent neutralization of the negative control (Mn.3 and Du156.12 for TAK and AMD, respectively). (C) CCR5 antagonist susceptibility was further assessed through a titration of the CCR5 antagonist maraviroc. No significant difference in CCR5 antagonist susceptibility was detected between variants isolated from plasma and milk (Wilcoxon test; P = 0.71). A CXCR4 antagonist titration was attempted, but 50% neutralization was not reached prior to AMD-induced cell toxicity.

FIG 5

Chemokine receptor usage by plasma and milk SIVsab variants. (A) SIVsab variant infectivity (TCID₅₀/ml) in NP-2 cells expressing CD4 and no chemokine receptor (parental), CCR5 and CXCR4, CCR5, CXCR4, or GPR15. SIVsab plasma and milk variants infected all cell lines with the exception of the parental cell line. Pseudovirus positive controls with known chemokine receptor usage included Du156.12/NL4.3ΔEnv_Luc (CCR5-tropic), Mn.3/NL4.3ΔEnv_Luc (CXCR4-tropic), 89.6P.18/NL4.3ΔEnv_Luc (CCR5, CXCR4, and CCR2 tropism), YU2 (CCR5 and CCR3 tropism), and YU2-6248wt (CCR5, CCR3, and GPR15 tropism). Gray shading indicates that infection was not detected. (B to E) Due to a lack of luminescence reporter genes in YU2-6248wt and YU2, Gag p24 concentration (pg/ml) was used to quantify infection by these viruses. Normalized infectivity (TCID₅₀/ng Gag p24) of SIVsab plasma and milk variants was similar in NP-2 cells expressing CD4 and CCR5 and CXCR4 (Wilcoxon test; P = 0.07) (B), CCR5 (Wilcoxon test; P = 0.54) (C), CXCR4 (Wilcoxon test; P = 0.14) (D), or GPR15 (Wilcoxon test; P = 0.21) (E). Comparison of infectivity across cell lines was not conducted due to variance in chemokine receptor expression levels.

FIG 6

Effect of type 1 IFNs on SIV infection and/or replication in human PBMCs. Increasing IFN-α (A) or IFN-β (B) concentrations on human PBMCs from two healthy donors (donors 1 and 2, indicated as circles and squares, respectively) inhibited productive infection by both the transmitter/founder SIVsab92018ivTF (blue symbols) (mean IC₅₀ = 85.4 pg/ml for IFN-α and mean IC₅₀ < 3.7 pg/ml for IFN-β) and SIVmac239 IMC (red symbols) (mean IC₅₀ = 16.8 pg/ml for IFN-α and mean IC₅₀ = 7.0 pg/ml for IFN-β) similarly.