Combined HIV-1 Envelope Systemic and Mucosal Immunization of Lactating Rhesus Monkeys Induces a Robust Immunoglobulin A Isotype B Cell Response in Breast Milk

Abstract

Maternal vaccination to induce anti-HIV immune factors in breast milk is a potential intervention to prevent postnatal HIV-1 mother-to-child transmission (MTCT). We previously demonstrated that immunization of lactating rhesus monkeys with a modified vaccinia Ankara (MVA) prime/intramuscular (i.m.) protein boost regimen induced functional IgG responses in milk, while MVA prime/intranasal (i.n.) boost induced robust milk Env-specific IgA responses. Yet, recent studies have suggested that prevention of postnatal MTCT may require both Env-specific IgA and functional IgG responses in milk. Thus, to investigate whether both responses could be elicited by a combined systemic/mucosal immunization strategy, animals previously immunized with the MVA prime/i.n. boost regimen received an i.n./i.m. combined C.1086 gp120 boost. Remarkably, high-magnitude Env-specific IgA responses were observed in milk, surpassing those in plasma. Furthermore, 29% of vaccine-elicited Env-specific B cells isolated from breast milk were IgA isotype, in stark contrast to the overwhelming predominance of IgG isotype Env-specific B cells in breast milk of chronically HIV-infected women. A clonal relationship was identified between Env-specific blood and breast milk B cells, suggesting trafficking of that cell population between the two compartments. Furthermore, IgA and IgG monoclonal antibodies isolated from Env-specific breast milk B cells demonstrated diverse Env epitope specificities and multiple effector functions, including tier 1 neutralization, antibody-dependent cellular cytotoxicity (ADCC), infected cell binding, and inhibition of viral attachment to epithelial cells. Thus, maternal i.n./i.m. combined immunization is a novel strategy to enhance protective Env-specific IgA in milk, which is subsequently transferred to the infant via breastfeeding.

Importance: Efforts to increase the availability of antiretroviral therapy to pregnant and breastfeeding women in resource-limited areas have proven remarkably successful at reducing HIV vertical transmission rates. However, more than 200,000 children are infected annually due to failures in therapy implementation, monitoring, and adherence, nearly half by postnatal HIV exposure via maternal breast milk. Intriguingly, in the absence of antiretroviral therapy, only 10% of breastfed infants born to HIV-infected mothers acquire the virus, suggesting the existence of naturally protective immune factors in milk. Enhancement of these protective immune factors through maternal vaccination will be a critical strategy to reduce the global pediatric AIDS epidemic. We have previously demonstrated that a high magnitude of HIV Env-specific IgA in milk correlates with reduced risk of infant HIV acquisition. In this study, we describe a novel HIV vaccine regimen that induces potent IgA responses in milk and therefore could potentially protect against breast milk HIV MTCT.

FIG 1

Combined i.n./i.m. HIV-1 Env vaccine regimen. Lactation was hormonally induced in four female rhesus macaques prior to weeks 0, 12, and 50 so that milk samples could be collected at each time point. Animals were primed with recombinant MVA expressing HIV Env C.1086 gp140 and then boosted twice with HIV Env C.1086 gp120 i.n. with R848 adjuvant. The animals were then boosted a third time using a combined i.n./i.m. Env boost with C.1086 gp120 i.n. plus R848 adjuvant as well as C.1086 gp120 i.m. plus STR8S-C adjuvant.

FIG 2

The combined i.n./i.m. HIV-1 Env boost enhances IgA responses in mammary and vaginal compartments over those elicited by i.n. boost alone. The gp120-specific IgG responses of animals given the MVA prime/i.n. boost regimen plus the combined i.m./i.n. boost are shown for plasma/milk (A) and vaginal/rectal secretions (C), while gp120-specific IgA responses are shown for plasma/milk (B) and vaginal/rectal secretions (D). Data points represent mean value for 4 animals, with standard deviations indicated by error bars. Plasma is in red, milk in blue, vaginal secretions in green, and rectal secretions in purple. The secretory IgA (SIgA) fraction of the gp120-specific IgA responses is in turquoise (B). IgA was not detectable in rectal secretions for any time point.

FIG 3

Isolation of C.1086 Env-specific IgA-producing B cells from breast milk of animal A6E030. (A and B) Env-specific B cells were isolated by selection of cells from blood (A) and milk (B) that are double positive for C.1086 Env labeled with two different fluorophores. (C and D) All MAbs isolated from Env-specific B cells in blood (C) and breast milk (D) were confirmed to be gp120 specific by ELISA. (E and F) While the blood gp120-specific MAbs were predominantly IgG isotype (E), those in milk were 29% IgA isotype (F). An identical analysis was performed for animal A6E088, though only 5 MAbs were isolated from plasma B cells and 5 MAbs from breast milk B cells.

FIG 4

Functional HIV-1 Env-specific antibody responses elicited in milk and plasma following combined i.n./i.m. Env immunization. (A and B) Neutralization responses against the tier 1 clade C virus MW965 in TZM-bl cells in plasma (A) and milk (B). Each colored line represents a different vaccinated animal. (C and D) ADCC activity against HIV-1 C.1086 gp120-coated CEM.NKR cells in plasma (C) and milk (D) following the combined i.n./i.m. Env boost. The dashed line indicates the starting dilution. Four of four animals had detectable ADCC activity in plasma, compared to zero of four animals with detectable activity in breast milk.

FIG 5

Clonal relationship of vaccine-elicited Env-specific blood and breast milk IgG isotype B cells. (A) A clonal lineage comprising monoclonal antibodies isolated from C.1086 Env-specific blood and breast milk B cells was identified in animal A6E030 following combined i.n./i.m. immunization. UCA, unmutated common ancestor. (B) Heavy-chain genes, HCDR3 length, and mutation frequency were determined for pairings using Cloanalyst.

FIG 6

Binding profile and determined specificities for 6 IgA and 6 IgG vaccine-elicited Env-specific MAbs isolated from breast milk B cells and selected for functional characterization. EC₅₀s (in μg/ml) obtained by ELISA are shown. Tested antigens are grouped into categories (Env glycoproteins, Env regional epitopes, CD4bs epitopes, and glycosylation mutants). NB, no binding detected.

FIG 7

Vaccine-elicited Env-specific MAbs from breast milk B cells bind infected cells and prevent viral binding to epithelial cells. (A) 6 MAbs (2 IgA and 4 IgG) demonstrated binding to the surface of C.1086 HIV-infected CEM.NKR_CCR5 cells. (B) 4 MAbs (2 IgA, 2 IgG) prevented C.MW965 from binding to GI epithelial cells. Dashed lines indicate the cutoff for positivity, set at a mean fluorescence intensity (MFI) of 100 for infected cell binding and a mean percent inhibition (MPI) of 31 (mean plus 2 SD for negative control) for inhibition of epithelial cell binding. The specificity of i.n./i.m. milk MAbs with a positive result is denoted above the error bars.

FIG 8

The ADCC effector function of MAbs is partially inhibited in milk following i.n./i.m. immunization. (A) Three MAbs were determined to mediate ADCC. DH527 (originally IgA, here made with a IgG backbone) has V2 specificity, DH532 is A32 blocking (C1 conformational), and DH535 is V3 specific. (B) Purified IgG from breast milk of animals A6E042 and A6E088 mediates a low level of ADCC activity exceeding that of whole milk from those same animals. (C) ADCC activity of ADCC-mediating monoclonal antibodies spiked into post-i.n./i.m. immunization milk is partially inhibited. (D) ADCC inhibition quantified by the percent change from MAbs spiked into prevaccination milk. The dotted lines indicate the threshold for positivity. Two experimental replicates were completed for panel A; panels B to D represent only a single experiment due to sample limitation.