Postnatally-transmitted HIV-1 Envelope variants have similar neutralization-sensitivity and function to that of nontransmitted breast milk variants

Abstract

Background: Breastfeeding is a leading cause of infant HIV-1 infection in the developing world, yet only a minority of infants exposed to HIV-1 via breastfeeding become infected. As a genetic bottleneck severely restricts the number of postnatally-transmitted variants, genetic or phenotypic properties of the virus Envelope (Env) could be important for the establishment of infant infection. We examined the efficiency of virologic functions required for initiation of infection in the gastrointestinal tract and the neutralization sensitivity of HIV-1 Env variants isolated from milk of three postnatally-transmitting mothers (n = 13 viruses), five clinically-matched nontransmitting mothers (n = 16 viruses), and seven postnatally-infected infants (n = 7 postnatally-transmitted/founder (T/F) viruses).

Results: There was no difference in the efficiency of epithelial cell interactions between Env virus variants from the breast milk of transmitting and nontransmitting mothers. Moreover, there was similar efficiency of DC-mediated trans-infection, CCR5-usage, target cell fusion, and infectivity between HIV-1 Env-pseudoviruses from nontransmitting mothers and postnatal T/F viruses. Milk Env-pseudoviruses were generally sensitive to neutralization by autologous maternal plasma and resistant to breast milk neutralization. Infant T/F Env-pseudoviruses were equally sensitive to neutralization by broadly-neutralizing monoclonal and polyclonal antibodies as compared to nontransmitted breast milk Env variants.

Conclusion: Postnatally-T/F Env variants do not appear to possess a superior ability to interact with and cross a mucosal barrier or an exceptional resistance to neutralization that define their capability to initiate infection across the infant gastrointestinal tract in the setting of preexisting maternal antibodies.

Figure 1

Phylogenetic comparison of virus env sequences from the plasma of postnatally-infected infants (red squares) and paired mother’s milk (blue circles). The infant of maternal subject 1209 (A) was first HIV DNA positive at 6 months of age, but a plasma sample was not available for virus sequencing until 12 months of age, thus infant virus evolution away from the transmitted maternal variant had occurred. The infant of maternal subject 4403 (B) was first HIV DNA positive at 3 months of age and infant plasma virus sequencing was performed from a sample collected at the same timepoint. The consensus of the infant plasma env sequences was confirmed to be identical to the maternal breast milk variant 4403 bmC5 by highlight plot (C). The postnatally-T/F variant from subject 4403 and the maternal milk env variant most closely related to the transmitted variant from subject 1209 are indicated with a red star.

Figure 2

No difference in binding to colonic epithelial cells or galactosyl ceramide (Galcer) between Env variants from women who did and did not postnatally transmit HIV-1 to their infants. Binding of milk Env variants from postnatal-transmitting (A) and nontransmitting (B) mothers to colonic epithelial HT-29 cells. (C) Binding of gp120 proteins expressed by milk Env virus variants to Galcer measured by biolayer interferometry. (D) Binding of gp120 variants from transmitting mothers to Galcer. Stars represent transmitted variants, error bars represent standard deviation and lines represent means of two experiments performed in triplicate.

Figure 3

DC transfer of breast milk HIV Env variants is at least partially Env mediated and is inhibited by lectin-binding molecules, including human lactoferrin. (A) Virions were incubated with a blocking agent (anti-Env monoclonal antibody, mannan, or lactoferrin) then incubated with mature DCs prior to inoculation of TZM-bl cells. DC transfer inhibition of a plasma Env virus variant from a transmitting mother by anti-HIV Env mAbs 2 G12 and CH08 (A) relative to a non-HIV-specific control antibody, Synagis (α-RSV) (B). Inhibition of DC transfer of a postnatally-transmitted infant-derived Env virus variant by α-methyl-D-mannopyranoside (C₇H₁₄0₆) and by human lactoferrin, relative to the respective uninhibited virus controls. Bars represent averages of three replicates and error bars represent standard deviations. Mean percent inhibition (M.P.I) values relative to the appropriate controls are indicated.

Figure 4

DC binding and transfer of breast milk and postnatally-acquired T/F infant Env variants. DC binding (A-C) and transfer (D-F) efficiencies of breast milk Env virus variants from transmitting (A, D) and nontransmitting (B, E) mothers and infant-derived Env virus variants (C,F). Bars represent the averages of three replicates from two independent experiments. The star represents the milk Env variant most closely related to the postnatally-transmitted breast milk Env variant in subject 1209.

Figure 5

No difference in the efficiency of viral entry between Env virus variants from the milk of transmitting and nontransmitting women, or postnatally infected infants. (A) Infectivity ratio (TCID₅₀/ml divided by p24 amount in ng/ml) of Env virus variants; (B) Inhibition of Env virus variant infectivity by CCR5 antagonist Maraviroc; (C) Inhibition of membrane fusion by entry inhibitor T20 peptide. Lines represent means IC50s (performed in duplicate for each virus) for each group of Env variants.

Figure 6

Neutralization sensitivity of Env variants from breast milk of postnatally transmitting or nontransmitting women and of infant T/F Env variants to broadly neutralizing polyclonal and monoclonal antibodies.