Breast milk transmission of HIV remains an important mode of infant HIV acquisition. Enhancement of mucosal HIV-specific immune responses in milk of HIV-infected mothers through vaccination may reduce milk virus load or protect against virus transmission in the infant gastrointestinal tract. However, the ability of HIV/SIV strategies to induce virus-specific immune responses in milk has not been studied. In this study, five uninfected, hormone-induced lactating, Mamu A*01+ female rhesus monkey were systemically primed and boosted with rDNA and the attenuated poxvirus vector, NYVAC, containing the SIVmac239 gag-pol and envelope genes. The monkeys were boosted a second time with a recombinant Adenovirus serotype 5 vector containing matching immunogens. The vaccine-elicited immunodominant epitope-specific CD8+ T lymphocyte response in milk was of similar or greater magnitude than that in blood and the vaginal tract but higher than that in the colon. Furthermore, the vaccine-elicited SIV Gag-specific CD4+ and CD8+ T lymphocyte polyfunctional cytokine responses were more robust in milk than in blood after each virus vector boost. Finally, SIV envelope-specific IgG responses were detected in milk of all monkeys after vaccination, whereas an SIV envelope-specific IgA response was only detected in one vaccinated monkey. Importantly, only limited and transient increases in the proportion of activated or CCR5-expressing CD4+ T lymphocytes in milk occurred after vaccination. Therefore, systemic DNA prime and virus vector boost of lactating rhesus monkeys elicits potent virus-specific cellular and humoral immune responses in milk and may warrant further investigation as a strategy to impede breast milk transmission of HIV.

HIV transmission via breast milk accounts for nearly half of the 350,000 new infant infections occurring annually in developing regions with high HIV prevalence (1). Although infant or maternal antiretroviral prophylaxis can reduce the incidence of breast milk HIV transmission (26), effective implementation of these long-term prophylaxis regimens will be complicated by infant toxicity, compliance, and inadequate health care infrastructure. Therefore, investigations of immunologic interventions, such as maternal or infant vaccines, to reduce postnatal transmission of HIV remain crucial for elimination of this mode of HIV transmission. Interestingly, in the absence of antiretroviral prophylaxis, only 10% of breastfeeding infants born to HIV-infected mothers will become infected postnatally (7). This low rate of HIV acquisition, despite daily low-dose exposure for up to 2 y, raises the possibility that protective immune responses in breast milk may prevent HIV acquisition in the majority of breastfeeding infants.

The risk for HIV transmission via breastfeeding has been associated with the level of HIV RNA and cell-associated HIV DNA in milk (812), in addition to maternal disease progression (8, 13, 14). Therefore, the reduction of cell-free and cell-associated virus load through enhancement of virus-specific immune responses in milk may result in reduced rates of HIV transmission via breast milk. HIV/SIV-specific CD8+ T lymphocyte responses are known to be critical for containment of systemic virus load (15) and have been identified in human and rhesus monkey milk (16, 17). Furthermore, recent studies of HIV/SIV virus evolution in milk suggest that the virus may replicate locally in the breast milk compartment (1820). Therefore, enhancement of the local HIV-specific CD8+ T lymphocyte responses in breast milk may improve containment of virus replication in the breast milk compartment and result in a decrease in breast milk virus load. In addition, passive postnatal administration of broadly HIV-neutralizing Ig protected infant macaques from HIV acquisition by oral exposure (21). Therefore, induction of potent, functional HIV-specific Ab responses in breast milk through maternal vaccination may play a role in blocking HIV acquisition in the infant gastrointestinal tract.

In this study, we aimed to establish the ability of a well-studied, systemic HIV/SIV vaccine prime-boost strategy to induce virus-specific cellular and humoral immune responses in breast milk of lactating monkeys. Nonhuman primates have proved to be an instrumental model for evaluating immunogenicity and efficacy of candidate HIV vaccines. Furthermore, we established a protocol for hormone-induction of lactation to investigate cellular and humoral immune responses in breast milk, because the breast milk produced by the hormone induction is immunologically similar to that of naturally lactating monkeys (17). It has long been noted that rDNA priming prior to virus vector boost enhances vaccine-elicited cellular immune responses (22); therefore, the monkeys in this study were primed with rDNA containing SIV gag, pol, and envelope immunogens. The monkeys were then boosted with matching SIV immunogens delivered via the attenuated poxvirus vector, NYVAC, a vector that induces strong polyfunctional CD4+ and CD8+ T lymphocyte responses (23), and replication-incompetent adenovirus vector serotype 5 (rAd5), a vector that induces potent cellular and humoral immune responses (2427). Adenovirus and poxvirus vectors were reported to induce mucosal CD8+ T lymphocyte responses in the gastrointestinal tract (28, 29), making these vectors excellent candidates for eliciting immune responses in breast milk.

Although induction of humoral immune responses in milk following systemic vaccination with polysaccharide, protein, or whole-cell vaccines and elicitation of cellular immune responses in milk following live-attenuated virus vaccination were reported (3037), it has not been established whether any candidate HIV vaccine can elicit humoral and/or cellular immune responses in breast milk. Therefore, our study aimed to characterize systemic cellular and humoral immune responses in breast milk following SIV vaccination. This detailed characterization of cellular and humoral immune responses in milk following systemic vaccination has implications for mother-to-child transmission of HIV and other neonatal pathogens transmitted via breast milk, such as human T lymphotrophic virus-1 and CMV. This study of breast milk immune responses elicited by a leading candidate HIV vaccine strategy will form the basis of future investigations of maternal vaccination to induce virus-specific immune responses that are protective against virus transmission via breastfeeding.

DNA plasmids containing the SIVmac239 gag-pol, and envelope genes, were generated by cloning the virus cDNA into the pVR1012 vector, as previously described (38, 39). Replication-incompetent, E1/E3-deleted rAd5 vectors expressing the SIVmac239 Gag and Envelope and SIV E660 Pol were prepared, as previously described (40). Finally, SIVmac239 gene-expressing NYVAC vectors were generated by the following protocol: plasmid transfer vectors pCyA20-SIVgag-pol and gp140 were constructed for insertion of the SIVmac239 gag-pol and envelope genes into the TK locus of the NYVAC genome. After the desired recombinant plasmids were isolated by screening for expression of β-galactosidase activity, BSC40 cells were infected with NYVAC virus (provided by Sanofi-Aventis, Paris, France) at a multiplicity of 0.05 PFU/cell and transfected with 10 μg DNA of the plasmid transfer vector using Lipofectamine reagent (Invitrogen, Carlsbad, CA). After 72 h, cells were harvested, sonicated, and used for recombinant virus screening. Recobinant NYVAC viruses containing the SIV genes and transiently coexpressing the β-gal marker gene were selected by four consecutive rounds of plaque purification in BSC40 cells stained with 5-bromo-4-chloro-3-indolyl β-galactoside (300 μg/ml). Next, recombinant NYVAC viruses containing the SIV genes and having the β-gal marker gene deleted through recombination at the TK site were isolated by three additional consecutive rounds of plaque purification, screening for nonstaining viral foci in BSC40 cells. Isolated plaques were grown and analyzed for the presence and expression of SIV gene products and for the absence of NYVAC-WT contamination by PCR and Western blot with SIV protein-specific mAbs (provided by Project EVA, European Union). Virus stocks were then generated in CEF cells and purified by two 45% sucrose gradient purifications.

Lactation was pharmacologically induced in five female Mamu-A*01+ rhesus monkeys using depot medroxyprogesterone and estradiol injections and an oral dopamine antagonist, as previously described (17). Once all five monkeys were lactating, the animals were vaccinated i.m. with 5 mg each rDNA plasmid containing the SIVmac239 gag-pol and envelope genes on weeks 0, 4, and 8. The monkeys were boosted i.m. and intradermally at week 28 with 108 PFU each of the NYVAC vectors containing the SIVmac239 gag-pol and envelope genes. The monkeys were boosted again at week 38 by i.m. administration of 1010 viral particles of each of the rAd5 vectors containing the SIVmac239 gag and envelope genes and SIV E660 pol gene. Milk (30–1000 μl) was collected two times per week throughout the vaccination schedule, and blood was collected weekly. Milk was separated into cellular, supernatant, and fat fractions by centrifugation, as previously described (17). Pinch biopsies from the colon and vaginal tract obtained at week 40 were digested with collagenase, and mononuclear cells were isolated on a Percoll gradient within 6 h, as previously described (41). Animals were maintained according to the “Guide for the Care and Use of Laboratory Animals.”

The vaccine-elicited CD8+ T lymphocyte response was measured by staining milk, colon, and vaginal cells and PBMCs with immunodominant Mamu A*01-restricted Env p54-allophycocyanin and Gag p11C-Q-Dot 655-labeled tetramers. Lymphocyte phenotype was assessed by anti–CD45-FITC (SP34.2), anti–CD3-Pacific Blue (SP34.2), anti–CD4-PerCP-Cy5.5 (L200), and anti–CD8-Alexa Fluor 700 (RPA-T8; all from BD Biosciences, San Jose, CA) mAb staining. Naive and memory lymphocyte subsets were defined by anti–CD95-allophycocyanin (DX2; BD Biosciences) and anti–CD28-PerCP-Cy5.5 (28.2; Beckman Coulter, Fullerton, CA) staining (naive: CD28+, CD95; effector memory: CD28, CD95+; central memory: CD28+, CD95+). The peak response was defined as the highest proportion of tetramer-staining CD8+ T lymphocytes measured after each vaccination (between 2 and 4 wk following each immunization for each monkey).

Activation status of breast milk CD4+ T lymphocytes after vaccination was assessed by mAb staining with anti–CD3-Pacific Blue (SP34.2), anti–CD4-Alexa Fluor 700 (RPA-T8), anti–CCR5-PE (3A9), anti–CD25-PECy7 (M-A251), and anti–HLA-DR-allophycocyanin-Cy7 [L243(G46-6), all from BD Biosciences]. Intracellular cytokine staining was performed after a 6-h exposure of PBMCs and breast milk cells to overlapping, protein-spanning pooled SIVman239 Gag peptides, as previously described (42), with the following additional Abs: anti–TNF-α–Pacific Blue (Mab11; eBiosciences, San Diego, CA), anti–IFN-γ–PE-Cy7 (B27; BD Biosciences), and anti–IL-2–allophycocyanin (MQ1-17H12; BD Biosciences). The proportion of cytokine-producing cells was determined by subtracting the proportion of unstimulated cytokine-producing cells from the proportion of Gag-stimulated cytokine-producing cells. An amine dye (Aqua Amine) was used to distinguish live from dead cells in all flow-cytometric analyses. Data were collected on the LSRII flow cytometer (BD Biosciences) with FACSdiva software and analyzed with FlowJo Software.

Automated complete cell counts of breast milk are skewed by the presence of fat droplets; therefore, they cannot be used to quantitate lymphocyte number. To calculate absolute lymphocytes per milliliter of milk and blood in a similar fashion, the absolute number of CD45+CD3+ lymphocytes quantitated by flow-cytometric analysis of each sample type was normalized to the ratio of fluorospheres added to the sample and quantitated by flow cytometry at the end of each staining procedure. To calculate the absolute lymphocyte count in blood, 100 μl EDTA-anticoagulated blood was mixed with 100 μl Flow-Count Fluorospheres (Beckman Coulter; 942 beads/μl) and stained with anti–CD45-FITC (DO58-1283) and anti–CD3-allophycocyanin-Cy7 (SP34.2; both from BD Biosciences). The RBCs were lysed and fixed using a TQ-Prep machine (Beckman Coulter), and the remaining blood cells and fluorospheres were washed, pelleted, and fixed. To calculate the absolute lymphocyte count in breast milk, 100 μl Flow-Count Fluorospheres were added to the total breast milk cell pellet. Breast milk cells were stained with the phenotyping Abs listed above. Sample data were collected within 4 h of fixation on the LSRII instrument (BD Biosciences) with FACSDiva software and analyzed with FlowJo software. After flow-cytometric analysis and quantitation of the fluorospheres that remained at the end of the procedure, the number of CD45+CD3+ lymphocyte events was normalized by the ratio of the number of fluorospheres added to the sample/the number of fluorospheres quantitated by flow cytometry. Finally, the normalized number of CD45+CD3+ lymphocytes was divided by the volume of the original sample. Absolute CD4+, CD8+, Gag p11c-specific CD8+, and Env p45-specific CD8+ T lymphocytes per milliliter were calculated by multiplying the percentage of the population by the normalized absolute CD45+CD3+ lymphocyte count. The peak response was defined as the highest absolute number of tetramer-staining CD8+ T lymphocytes measured after each vaccination (between 2 and 4 wk following each immunization for each monkey).

SIV Envelope-binding IgG and IgA were measured by incubation of serial 3-fold dilutions of plasma and milk supernatant in duplicate in a 96-well plate coated with rSIVmac239 gp130 (ImmunoDiagnostics, Woburn, MA). After blocking with PBS with 5% nonfat dried milk and 10% FBS, SIV Envelope-binding Ab was detected by an HRP-conjugated, polyclonal goat anti-monkey IgG (Alpha Diagnostics, Owings Mills, MD) or anti-monkey IgA (Rockland, Gilbertsville, PA) Ab and the addition of ABTS-2 peroxidase substrate system (Kirkegaard & Perry Laboratories, Gaithersburg, MD). OD was measured at 450 nm. SIV envelope-specific Ab titer was calculated as the inverse of the lowest dilution of plasma or milk supernatant that had an average OD that was 2-fold greater than that of the PBS negative control.

To measure neutralizing Ab titer in blood and milk, a stock of molecularly cloned T cell line-adapted (TCLA) SIVmac239 Envelope-pseudotyped virus was prepared by transfection in 293T cells and titrated in TZM.bl, cells as previously described (43). Neutralization was measured by reduction in luciferase reporter gene expression after a single round of infection in TZM.bl cells, as previously described (43). Briefly, 200 tissue culture infectious dose 50% of virus was incubated with 3-fold serial dilutions of plasma or milk supernatant in duplicate for 1 h at 37°C in 96-well flat-bottom culture plates. TZM.bl cells were added (1 × 104/well in 100 μl volume) in 10% DMEM growth medium containing DEAE-Dextran (Sigma-Aldrich, St. Louis, MO) at a final concentration of 11 μg/ml. Assay controls included replicate wells of TZM.bl cells alone (cell control) and TZM.bl cells with virus (virus control). Following a 48-h incubation at 37°C, 150 μl assay medium was removed from each well, and 100 μl Bright-Glo luciferase reagent (Promega, Madison, WI) was added. The cells were allowed to lyse for 2 min, then 150 μl the cell lysate was transferred to a 96-well black solid plate, and luminescence was measured. The ID50 titer was calculated as the plasma dilution that caused a 50% reduction in relative luminescence units compared with the virus control wells after subtraction of cell control relative luminescence units.

All comparisons of the magnitude of the vaccine-elicited cellular or humoral immune responses in blood, milk, and other mucosal compartments were performed using the paired, nonparametric Wilcoxon signed-rank test with Prism software (GraphPad, San Diego, CA). The lowest obtainable p value using this paired, nonparametric test with n = 5 animals is p = 0.06.

Five Mamu A*01+ hormone-induced lactating female rhesus monkeys received priming immunizations i.m. with 5 mg of rDNA plasmids with SIVmac239 gag-pol and env gene inserts on weeks 0, 4, and 8. Animals were boosted i.m. and intradermally at week 28 with 108 PFU of recombinant attenuated pox vector, NYVAC, containing SIVmac239 gag-pol and env genes. Finally, animals were repeat boosted i.m. at week 38 with 1010 viral particles of recombinant adenovirus serotype 5 containing SIV gag, pol, and envelope. The vaccine-elicited cellular and humoral immune responses were assessed in blood and breast milk following each immunization. Animals underwent colon and vaginal biopsy 2 wk following the final immunization to compare the magnitude of the cellular immune responses in breast milk with these mucosal compartments (Fig. 1).

FIGURE 1.

Study design and vaccine regimen in lactating rhesus monkeys. Five lactating rhesus monkeys were primed with rDNA expressing SIVmac239 Gag, Pol, and Env at 0, 4, and 8 wk. Twenty weeks later, the monkeys were boosted with NYVAC expressing SIVmac239 Gag-Pol and Env. Finally, 10 wk after NYVAC boost, animals were boosted again with rAd5 expressing SIV Gag, Pol, and Env. CD8+ tetramer staining was performed on milk and blood weekly, and staining for activation molecules was performed on milk weekly. Intracellular cytokine staining was performed 3 wk after each vaccine type. Colon and vaginal biopsies were performed 2 wk following rAd5 boost.

FIGURE 1.

Study design and vaccine regimen in lactating rhesus monkeys. Five lactating rhesus monkeys were primed with rDNA expressing SIVmac239 Gag, Pol, and Env at 0, 4, and 8 wk. Twenty weeks later, the monkeys were boosted with NYVAC expressing SIVmac239 Gag-Pol and Env. Finally, 10 wk after NYVAC boost, animals were boosted again with rAd5 expressing SIV Gag, Pol, and Env. CD8+ tetramer staining was performed on milk and blood weekly, and staining for activation molecules was performed on milk weekly. Intracellular cytokine staining was performed 3 wk after each vaccine type. Colon and vaginal biopsies were performed 2 wk following rAd5 boost.

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We first investigated the changes in lymphocyte number and relative proportions of CD4+ and CD8+ T memory and naive lymphocytes in milk and blood weekly following systemic vaccination. The lymphocyte number and the proportion of CD8+ effector memory T lymphocytes increased greatly following SIV infection (17), but changes in lymphocyte number and phenotype in milk following maternal vaccination have not been studied. The absolute number of lymphocytes in breast milk before vaccination (median, 4300 CD3+ T cells/ml; range: 1,400–41,800) did not trend toward a statistically significant difference at the peak of the response after each immunization (data not shown). Furthermore, the proportion of CD4+ (median, 64.3%; range: 30–72.4%) or CD8+ (median, 32.5%; range: 25.8–72.8%) T lymphocytes in milk prior to vaccination did not change following each systemic immunization (data not shown). Finally, the proportion of effector memory (median, 89.3%; range: 84.6–96.2%), central memory (median, 10.7%; range: 3.7–15.4%), or naive (median, 0%; range: 0–1.8%) CD4+ T lymphocytes and effector memory (median, 41.9%; range: 33.3–70.2%), central memory (median, 58.1%; range: 30.3–66.7%), or naive (median, 0%; range 0%) CD8+ T lymphocytes did not significantly change in milk following each systemic immunization (data not shown). Accordingly, there were no changes in the number and proportion of memory and naive CD4+ and CD8+ T lymphocytes in blood following vaccination (data not shown).

The vaccine-elicited CD8+ T lymphocyte response specific for the immunodominant Mamu A*01-restricted epitope, Gag p11C, and the subdominant Mamu A*01-restricted epitope, Env p54, were assessed weekly following each immunization. The peak proportion of CD8+ T lymphocytes that were specific for the Gag p11C epitope was greater in breast milk (median, 2.9%; range: 1.8–3.7%) compared with blood (median, 0.41%; range: 0.21–0.87%) following systemic rDNA immunization in all animals (p = 0.06). Upon systemic boosting with the SIV NYVAC live viral vectors, the peak proportion of Gag p11C-specific CD8+ T lymphocytes in breast milk (median, 1.6%; range: 1.3–3.7%) was similar in magnitude to that in blood (median, 1.1%; range: 0.7–2.0%) (p = 0.19). Finally, the median and range of the peak proportion of Gag p11C-specific CD8+ T lymphocytes were higher in milk (median, 5.4%; range: 2.5–9.1%) than in blood (median, 3.5%; range: 2.0–5.1%) following the rAd5 boost, but the responses in each compartment were not different enough to approach statistical significance (p = 0.31) (Fig. 2A, 2B). Despite the higher-magnitude Gag p11C-specific CD8+ T lymphocyte response in breast milk, the median absolute number of Gag p11C-specific CD8+ T lymphocytes remained one to two logs lower in milk than in the blood throughout the immunization schedule as a result of the low lymphocyte number in breast milk (p = 0.06 for all comparisons of blood and milk absolute Gag p11C-specific CD8+ T lymphocyte number after each immunization) (Fig. 2C, 2D).

FIGURE 2.

Robust vaccine-elicited CD8+ T lymphocyte responses appear in breast milk following rDNA, pox vector, and recombinant adenovirus vector immunization. Peak Gag p11C-specific CD8+ T lymphocyte proportion in blood (A) and milk (B) following DNA prime and NYVAC and rAd5 boost. Peak absolute number of Gag p11C-specific CD8+ T lymphocytes/ml in blood (C) and milk (D) following each immunization. Peak Env p54-specific CD8+ T lymphocyte proportion in blood (E) and milk (F) following DNA prime and NYVAC and rAd5 boost. Peak absolute number of Env p54-specific CD8+ T lymphocytes/ml in blood (G) and milk (H) following each immunization.

FIGURE 2.

Robust vaccine-elicited CD8+ T lymphocyte responses appear in breast milk following rDNA, pox vector, and recombinant adenovirus vector immunization. Peak Gag p11C-specific CD8+ T lymphocyte proportion in blood (A) and milk (B) following DNA prime and NYVAC and rAd5 boost. Peak absolute number of Gag p11C-specific CD8+ T lymphocytes/ml in blood (C) and milk (D) following each immunization. Peak Env p54-specific CD8+ T lymphocyte proportion in blood (E) and milk (F) following DNA prime and NYVAC and rAd5 boost. Peak absolute number of Env p54-specific CD8+ T lymphocytes/ml in blood (G) and milk (H) following each immunization.

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The vaccine-elicited CD8+ T lymphocyte response specific for the subdominant Env p54 epitope was of greater magnitude in milk than in blood after rDNA (milk median, 0.9%; range: 0.7–2.9%; blood median, 0.2%; range: 0.1–0.3%), NYVAC (milk median, 0.7%; range: 0.5–2.2%; blood median, 0.2%; range: 0.1–0.5%), and rAd5 (milk median, 2.3%; range: 1.4–5.9%; blood median, 0.1%; range: 0.1–0.2%) (all p = 0.06) (Fig. 2E, 2F). In contrast to the vaccine-elicited immunodominant Gag p11C-specific CD8+ T lymphocyte number in milk, the median absolute number of vaccine-elicited Env p54-specific CD8+ T lymphocytes in milk was similar to that in blood after rDNA (milk median, 120 cells/ml; range: 15–1380 cells/ml; blood median, 750 cells/ml; range: 456–2334 cells/ml), and rAd5 (milk median, 266 cells/ml; range: 30–1131 cells/ml; blood median, 985 cells/ml; range: 312–1110 cells/ml) immunization (p = 0.31 and 0.19). However, the absolute number of vaccine-elicited Env p54-specific CD8+ T lymphocytes did trend toward being significantly lower in milk (median, 84 cells/ml; range: 22–269 cells/ml) than in blood (median, 1727 cells/ml; range: 606–4319 cells/ml) after NYVAC immunization (p = 0.06), again because of the low cell number in milk (Fig. 2G, 2H). The peak median proportion and absolute number of vaccine-elicited, virus-specific CD8+ T cells after each prime and boost are summarized in Table I.

Table I.
Median vaccine-elicited cellular and humoral immune responses in blood and milk of systemically vaccinated, lactating rhesus monkeys
Immune ParameterVaccinationBloodMilk
Gag p11C-specific CD8+ T cells (%) Prevaccination 0.01 
 rDNA 0.4 2.9 
 NYVAC 1.1 1.7 
 rAd5 3.5 7.3 
Abs Gag p11C-specific CD8+ T cells (cells/ml) Prevaccination 25 
 rDNA 1,600 391 
 NYVAC 10,900 93 
 rAd5 18,700 873 
Env p54-specific CD8+ T cells (%) Prevaccination 
 rDNA 0.2 0.9 
 NYVAC 0.24 0.7 
 rAd5 0.1 2.3 
Abs Env p54-specific CD8+ T cells (cell/ml) Prevaccination 
 rDNA 749 119 
 NYVAC 1,727 84 
 rAd5 985 266 
TCLA SIV-neutralizing Ab (ID50 titer) Prevaccination 13 11 
 rDNA 1,047 27 
 NYVAC 1,995 24 
 rAd5 10,422 107 
Anti-SIV gp130 IgG (titer) Prevaccination 
 rDNA 300 
 NYVAC 10,000 
 rAd5 10 10,000 
Anti-SIV gp130 IgA (titer) Prevaccination 
 rDNA 
 NYVAC 65 
 rAd5 30 
Immune ParameterVaccinationBloodMilk
Gag p11C-specific CD8+ T cells (%) Prevaccination 0.01 
 rDNA 0.4 2.9 
 NYVAC 1.1 1.7 
 rAd5 3.5 7.3 
Abs Gag p11C-specific CD8+ T cells (cells/ml) Prevaccination 25 
 rDNA 1,600 391 
 NYVAC 10,900 93 
 rAd5 18,700 873 
Env p54-specific CD8+ T cells (%) Prevaccination 
 rDNA 0.2 0.9 
 NYVAC 0.24 0.7 
 rAd5 0.1 2.3 
Abs Env p54-specific CD8+ T cells (cell/ml) Prevaccination 
 rDNA 749 119 
 NYVAC 1,727 84 
 rAd5 985 266 
TCLA SIV-neutralizing Ab (ID50 titer) Prevaccination 13 11 
 rDNA 1,047 27 
 NYVAC 1,995 24 
 rAd5 10,422 107 
Anti-SIV gp130 IgG (titer) Prevaccination 
 rDNA 300 
 NYVAC 10,000 
 rAd5 10 10,000 
Anti-SIV gp130 IgA (titer) Prevaccination 
 rDNA 
 NYVAC 65 
 rAd5 30 

We were able to demonstrate SIV Gag-specific IFN-γ, TNF-α, and IL-2 production in CD4+ and CD8+ T lymphocytes in breast milk by intracellular cytokine staining 3 wk following rDNA priming, as well as NYVAC and rAd5 boost in two vaccinated monkeys with adequate lymphocyte number (CD3+ T lymphocyte number > 1000 per condition) (Fig. 3). The proportion of Gag-specific cytokine-producing CD4+ T lymphocytes in milk was similar to that in blood following rDNA prime. However, the proportion of Gag-specific, IFN-γ–producing CD4+ T lymphocytes was ≥5-fold higher in milk than in blood in both animals following NYVAC immunization. In addition, the proportions of Gag-specific TNF-α and IL-2–producing CD4+ T lymphocytes were ≥5-fold higher in milk than in blood in one animal and were similar or greater in magnitude in the other animal following NYVAC. Finally, the proportion of Gag-specific IFN-γ–, TNF-α–, and IL-2–producing CD4+ T lymphocytes was ≥4-fold higher in milk than in blood of both animals following rAd5 vaccination. As expected, the magnitude of the vaccine-elicited CD4+ T lymphocyte responses was higher in blood and milk following NYVAC boost compared with those elicited by rAd5 boost, because poxvirus vectors are known to elicit strong CD4+ T lymphocyte responses (44) (Fig. 4A, 4B).

FIGURE 3.

Vaccine-elicited, cytokine-producing CD8+ T lymphocyte responses in breast milk following systemic vaccination. A, Gating strategy used to analyze intracellular cytokine production of milk CD4+ and CD8+ T lymphocytes. B, Representative dot plots of intracellular cytokine staining in unstimulated, Gag peptide, and Staphylococcus endotoxin B (SEB)-stimulated blood and milk CD8+ T lymphocytes after rAd5 boost in one animal (294). Total milk CD8+ T cell number analyzed in this monkey was 1228 (unstimulated), 1184 (Gag peptide stimulated), and 906 (SEB stimulated).

FIGURE 3.

Vaccine-elicited, cytokine-producing CD8+ T lymphocyte responses in breast milk following systemic vaccination. A, Gating strategy used to analyze intracellular cytokine production of milk CD4+ and CD8+ T lymphocytes. B, Representative dot plots of intracellular cytokine staining in unstimulated, Gag peptide, and Staphylococcus endotoxin B (SEB)-stimulated blood and milk CD8+ T lymphocytes after rAd5 boost in one animal (294). Total milk CD8+ T cell number analyzed in this monkey was 1228 (unstimulated), 1184 (Gag peptide stimulated), and 906 (SEB stimulated).

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FIGURE 4.

The vaccine-elicited CD4+ and CD8+ T lymphocyte responses in milk are of higher magnitude and display a distinct polyfunctional cytokine profile compared with that in blood following NYVAC and rAd5 boost. The proportion of blood (A) and milk (B) CD4+ T lymphocytes and blood (C) and milk (D) CD8+ T lymphocytes producing IFN-γ, TNF-α, and IL-2 3 wk following the third rDNA prime and NYVAC and rAd5 boost in two monkeys (294 and 402) with adequate breast milk cell number for this analysis. The polyfunctional cytokine profile of vaccine-elicited, Gag-specific CD4+ T lymphocytes in blood (E) and milk (F) and CD8+ T lymphocytes in blood (G) and milk (H) 3 wk following rAd5 boost in the two monkeys (294 and 402) with adequate breast milk cell number for the analysis.

FIGURE 4.

The vaccine-elicited CD4+ and CD8+ T lymphocyte responses in milk are of higher magnitude and display a distinct polyfunctional cytokine profile compared with that in blood following NYVAC and rAd5 boost. The proportion of blood (A) and milk (B) CD4+ T lymphocytes and blood (C) and milk (D) CD8+ T lymphocytes producing IFN-γ, TNF-α, and IL-2 3 wk following the third rDNA prime and NYVAC and rAd5 boost in two monkeys (294 and 402) with adequate breast milk cell number for this analysis. The polyfunctional cytokine profile of vaccine-elicited, Gag-specific CD4+ T lymphocytes in blood (E) and milk (F) and CD8+ T lymphocytes in blood (G) and milk (H) 3 wk following rAd5 boost in the two monkeys (294 and 402) with adequate breast milk cell number for the analysis.

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The proportions of Gag-specific cytokine-producing CD8+ T lymphocytes in milk were similar or higher than that in blood after rDNA priming and NYVAC and rAd5 boost. Of note, the proportions of Gag-specific TNF-α– and IL-2–producing CD8+ T lymphocytes in milk were ≥1 log higher than that in blood in both monkeys following rAd5 boost. Furthermore, the proportion of Gag-specific IFN-γ–producing CD8+ T lymphocytes was 1-log higher in milk of one animal than that in blood following rAd5 boost. As expected, the proportions of Gag-specific TNF-α– and IL-2–producing CD8+ T lymphocytes in milk were higher in both monkeys following rAd5 boost than those following rDNA prime and NYVAC boost, because rAd5 vectors are known to induce strong mucosal and systemic CD8+ T lymphocyte responses (23, 24, 40) (Fig. 4C, 4D).

We then compared the polyfunctional cytokine profile of vaccine-elicited breast milk lymphocytes to that in blood after each vaccine vector boost. Interestingly, a distinct polyfunctional cytokine-production profile of the vaccine-elicited T lymphocyte was elicited in milk compared with that in blood (Fig. 4E–H). Although Gag-specific IFN-γ only-producing cells were a predominant population in blood and milk CD4+ and CD8+ T lymphocyte subsets, the TNF-α– and IFN-γ–producing cells were predominant only in milk vaccine-elicited CD4+ and CD8+ T lymphocytes. Furthermore, the TNF only-producing cells and IL-2, TNF-, and IFN-producing populations accounted for a higher proportion of cytokine-producing cells in milk CD4+ and CD8+ T lymphocytes than in the blood (Fig. 4E–H). This same pattern was found after NYVAC and rAd5 vector boost. This difference in the polyfunctional cytokine profile of vaccine-elicited T lymphocytes in milk and blood likely reflects the regulated trafficking of activated lymphocytes into the breast milk compartment or local proliferation of memory lymphocytes in milk.

We compared the proportion of Gag p11C-specific CD8+ T lymphocytes in blood, milk, colon, and vaginal tract 2 wk following the rAd5 boost. The median proportion of vaccine-elicited CD8+ T lymphocytes specific for the immunodominant Gag p11C epitope in milk (median, 5.4%; range: 2.5–9.1%) was higher than that in colon (median, 2.2%; range: 0.7–2.9%) and trended toward a statistically significantly different (p = 0.12). In contrast, the median proportion of vaccine-elicited CD8+ T lymphocytes specific for Gag p11C epitope was similar in the milk and the vaginal tract (median, 3.1%; range: 0.8–8.6%) (p = 0.44) (Fig. 5). Therefore, the cellular immune responses elicited by systemic DNA prime-virus vector boost in milk were of similar or greater magnitude compared with that elicited in other mucosal compartments.

FIGURE 5.

The peak proportion of vaccine-elicited Gag p11C-specific CD8+ T lymphocytes in milk is higher than that in colon and equivalent to that in the vaginal tract following rAd5 boost. The proportion of Gag p11C-specific CD8+ T lymphocytes in blood, milk, colon, and vagina 2 wk after rAd5 boost.

FIGURE 5.

The peak proportion of vaccine-elicited Gag p11C-specific CD8+ T lymphocytes in milk is higher than that in colon and equivalent to that in the vaginal tract following rAd5 boost. The proportion of Gag p11C-specific CD8+ T lymphocytes in blood, milk, colon, and vagina 2 wk after rAd5 boost.

Close modal

Vaccine-elicited neutralizing Ab responses were detected in milk against a TCLA SIVmac239 in a pseudovirus TZM-bl–neutralization assay after NYVAC and rAd5 boost (Fig. 6B). However, this neutralizing Ab response in milk (median ID50, 107; range: 63–966) was ≥2 logs lower than that in plasma (median ID50, 10,442; range: 7.350–21,870) (p = 0.06) (Fig. 6A). An SIV envelope-binding IgG response was detected in milk after NYVAC (median titer, 3; range: 3–100) and rAd5 (median titer, 10; range: 3–100) boost, but the titer was 2–3 logs lower than that in plasma (post-NYVAC median titer, 10,000; range: 3,000–30,000; post-rAd5 median titer, 10,000; range: 10,000–30,000) (p = 0.06) (Fig. 6C, 6D). Furthermore, an SIV Envelope-binding IgA response was only detected in milk of one animal following NYVAC boost and in no animal after rAd5 boost (Fig. 6F). This limited detection of SIV Envelope-binding IgA response in milk is in contrast to the systemic SIV Envelope-binding IgA response that was detected in all animals after NYVAC (median titer, 30; range: 30–300) and rAd5 (median titer, 30; range: 30–100) boost (Fig. 6E). Therefore, the vaccine-elicited neutralizing Ab response in milk is likely attributable to the SIV envelope-specific IgG in milk rather than SIV envelope-specific secretory IgA. The median SIV envelope-binding and -neutralizing Ab responses after each vaccine vector are summarized in Table I.

FIGURE 6.

Limited SIV-neutralizing and SIV envelope-binding mucosal IgA response in breast milk following DNA prime and live virus vector boost. TCLA SIV-neutralizing Ab response in plasma (A) and milk (B) following DNA prime and NYVAC and rAd5 boost. SIVmac239 gp130-binding IgG response in plasma (C) and milk (D) following DNA prime and NYVAC and rAd5 boost. SIVmac239 gp130-binding IgA response in plasma (E) and milk (F) following DNA prime and NYVAC and rAd5 boost.

FIGURE 6.

Limited SIV-neutralizing and SIV envelope-binding mucosal IgA response in breast milk following DNA prime and live virus vector boost. TCLA SIV-neutralizing Ab response in plasma (A) and milk (B) following DNA prime and NYVAC and rAd5 boost. SIVmac239 gp130-binding IgG response in plasma (C) and milk (D) following DNA prime and NYVAC and rAd5 boost. SIVmac239 gp130-binding IgA response in plasma (E) and milk (F) following DNA prime and NYVAC and rAd5 boost.

Close modal

Because activation of CD4+ T lymphocytes following vaccination has been raised as a concern for enhancing virus transmission (45, 46), we investigated the activation status of the CD4+ T lymphocytes in milk by assessing their IL-2R (CD25), MHC class II molecule HLA-DR, and the HIV coreceptor CCR5 expression throughout the immunization schedule. There was a 2–10% increase in the proportion of CD4+ T lymphocytes in milk expressing CCR5 following the second and third systemic rDNA prime, but this increase lasted ≤2 wk. Because the median number of CD4+ T lymphocytes in milk during the vaccination schedule was 2752 cells/ml, the maximum absolute increase in CCR5-expressing CD4+ T lymphocytes following DNA vaccination is 275 cells/ml. There was no apparent increase in CCR5 expression following NYVAC boost; however, there was a 1–3% increase in the proportion of CCR5-expressing cells lasting ≤2 wk following rAd5 boost (Fig. 7A), representing a maximum absolute increase in CCR5-expressing CD4+ T lymphocytes of 82 cells/ml. Similarly, the proportion of milk CD4+ T lymphocytes expressing CD25 increased by 1–5% following the second and third rDNA prime, but again this increase lasted ≤2 wk. This increased proportion of CD25-expressing CD4+ T lymphocytes following DNA priming represents a maximum absolute increase of 137 CD25-expressing CD4+ T cells/ml. There was a minimal increase in the proportion of CD25-expressing CD4+ T lymphocytes following NYVAC and rAd5 boost (Fig. 7B). Similarly, the proportion of CD4+ T lymphocytes expressing HLA-DR increased 5–10% following the second and third rDNA prime, but this increase lasted <2 wk and represents a maximum absolute increase of 275 HLA-DR–expressing CD4+ T lymphocytes/ml. Finally, there was a small increase in the proportion of CD4+ T lymphocytes expressing HLA-DR in only one of five monkeys following NYVAC boost and in two of five monkeys following rAd5 boost that lasted <2 wk (Fig. 7C). Furthermore, there was no significant change in the mean fluorescence intensity of the CCR5-, CD25-, or HLA-DR–expressing cells after vaccination to indicate a higher level of activation-molecule expression (data not shown). Therefore, the activation of breast milk CD4+ T lymphocytes after systemic vaccination composed of rDNA prime and live virus vector boost was minimal and self-limited.

FIGURE 7.

Limited and transient activation of CD4+ T lymphocytes in milk following systemic DNA prime and NYVAC and rAd5 boost. The proportion of CD4+ T lymphocytes in milk expressing CCR5 (A), IL-2R CD25 (B), and the HLA-DR MHC class II molecule (C) following systemic DNA prime and NYVAC and rAd5 boost.

FIGURE 7.

Limited and transient activation of CD4+ T lymphocytes in milk following systemic DNA prime and NYVAC and rAd5 boost. The proportion of CD4+ T lymphocytes in milk expressing CCR5 (A), IL-2R CD25 (B), and the HLA-DR MHC class II molecule (C) following systemic DNA prime and NYVAC and rAd5 boost.

Close modal

In this study, we established that systemic rDNA priming and virus vector boost can elicit robust immunogen-specific, polyfunctional CD4+ and CD8+ T lymphocyte responses in breast milk. Interestingly, the polyfunctional cytokine-production profile of immunogen-specific breast milk lymphocytes was distinct from that in the blood. This finding likely reflects the regulated trafficking of vaccine-elicited memory lymphocytes into this mucosal compartment or local proliferation of this immunogen-specific lymphocyte population within the breast milk compartment. These vaccine-elicited, virus-specific breast milk lymphocytes may be active in the maternal breast milk compartment and could enhance containment of viruses that are shed in breast milk, such as HIV and CMV. The vaccine-elicited breast milk lymphocytes could also be active in the infant gastrointestinal tract, and these responses could play a role in the prevention of mucosally transmitted neonatal pathogens.

We previously described a robust virus-specific CD8+ T lymphocyte response in breast milk during acute SIV infection that was two to three times higher than that in blood (17). Furthermore, studies of HIV/SIV evolution in breast milk suggested that the breast milk virus population is at least partially populated by virus produced by locally infected cells, reflected by the frequent occurrence of groups of genetically identical viruses in breast milk that are absent or less common in plasma (1820). Moreover, we reported that breast milk virus quasispecies escape MHC class I-restricted immunodominant CTL responses by mutation of the restricted epitopes at a faster or similar rate to that of the blood virus population (17). Therefore, virus replicating in the breast milk compartment is likely evolving in response to immune pressure from virus-specific CD8+ T lymphocytes. Collectively, these studies suggest that virus-specific CD8+ T lymphocytes act on locally replicating virus in the breast milk compartment. Thus, enhancement of the virus-specific CD8+ T lymphocyte responses in breast milk through maternal vaccination may contribute to improved control of virus replication in the breast milk compartment. Reduction of breast milk virus load may result in a reduction of infant virus transmission.

In this study, we noted high-magnitude Gag-specific CD4+ T lymphocyte responses in milk and blood following NYVAC boost, consistent with the robust CD4+ T lymphocyte responses elicited by poxvirus vectors (44). High-magnitude CD8+ T lymphocyte responses and neutralizing Ab responses in milk and blood were generated following rAd5 boost, consistent with previous reports of vaccine-elicited rAd5 responses (2427). However, the enhanced CD8+ T lymphocyte and neutralizing Ab responses following rAd5 boost may be an additive effect of a second boost administered over a short time interval. Therefore, elicitation of durable, high-magnitude breast milk virus-specific CD8+ T lymphocyte responses for containment of breast milk virus replication and neutralizing Ab responses with the ability to block virus transmission in the infant gastrointestinal tract may require more than one vector boost immunization.

The humoral immune responses elicited in breast milk by this vaccine regimen were low titer and consisted of only IgG responses. Although this limited virus-specific IgA response mirrors the HIV/SIV-specific Ab profile in breast milk of chronically infected hosts (17, 4749), the predominant breast milk Ab isotype is secretory IgA. Induction of an SIV Envelope-specific IgA response in breast milk may play a role in blocking virus transmission in the infant gastrointestinal tract. The vaccine regimen investigated in this study targets cellular immune responses and, therefore, is not expected to generate potent mucosal Ab responses. It is possible that an Ab-based vaccine regimen, such as a virus vector prime and recombinant protein boost similar to the regimen that elicited a potentially protective Ab response in the recent Thai trial (50), may be more likely to elicit a mucosal IgA response in breast milk (51). Furthermore, using a mucosal route of immunization may be more effective at inducing a mucosal IgA response in breast milk (5153).

Finally, vaccine-induced activation of CD4+ T lymphocytes has been suggested as a hypothesis for the cause of increased HIV infections in the vaccine arm of the Step (HIV Vaccine Trials Network) trial evaluating a DNA prime/recombinant adenovirus vector boost HIV vaccine strategy (45, 46). Therefore, we sought to assess the extent of the activation of CD4+ T lymphocytes in milk following systemic vaccination in uninfected monkeys. Activated CD4+ T lymphocytes in milk of HIV-infected women may be more apt at producing virus and interacting with the breastfeeding infant’s gastrointestinal tract. We found minimal and transient increases in the proportion of CD4+ T lymphocytes in milk expressing molecules of activation and the HIV/SIV coreceptor CCR5 following vaccination in these uninfected, lactating monkeys. A similar pattern of limited and transient activation occurred in systemic and colonic CD4+ T lymphocytes following similar vaccine regimens (41). It is unlikely that these transient and minimal increases in the proportion of activated CD4+ T lymphocytes following vaccination would be clinically significant in HIV-infected lactating women, because breast milk lymphocytes are already highly activated in this setting (54).

The demonstration of vaccine-elicited cellular and humoral immune responses following systemic administration of a candidate HIV/SIV vaccine regimen provides a platform for further development of maternal vaccine strategies that may impede breast milk transmission of HIV. Although antiretroviral prophylaxis will be a mainstay of prevention of HIV transmission via breastfeeding, vaccination could provide a safe and durable adjunctive mechanism of preventing breast milk transmission of HIV. Vaccine strategies are less reliant on patient compliance and health care infrastructure than are drug interventions. Furthermore, an effective maternal vaccine administered after delivery would avoid fetal and infant toxicities. Therefore, it is important to continue to evaluate vaccine regimens well-suited for induction of mucosal responses in breast milk and assess the efficacy of these interventions in protection against HIV/SIV transmission via breastfeeding in nonhuman primate models (55) and clinical studies.

We thank Gary Nabel and the Vaccine Research Center (National Institutes of Health, Bethesda, MD) for provision of the SIVmac 239 rDNA vaccine constructs. Furthermore, we thank Michelle Lifton, Keith Reimann, and James Whitney for advice and technical assistance.

Disclosures The authors have no financial conflicts of interest.

This work was supported by National Institutes of Health Grant K08AI087992 (to S.R.P.), the Center for HIV/AIDS Vaccine Immunology (R01AI067854; to S.R.P. and N.L.L.), the Children’s Hospital Boston Faculty Development Award (to S.R.P.), the Pediatric Infectious Disease Society/St. Jude Children’s Hospital Basic Science Research Award (to S.R.P.), and the Bill and Melinda Gates Collaboration for AIDS Vaccine Discovery Vaccine Immune Monitoring Consortium Grant 38619 (to M.S.S.).

Abbreviations used in this paper:

rAd5

replication-incompetent adenovirus vector serotype 5

SEB

Staphylococcus endotoxin B

TCLA

T cell line adapted.

1
Nduati
R.
,
John
G.
,
Mbori-Ngacha
D.
,
Richardson
B.
,
Overbaugh
J.
,
Mwatha
A.
,
Ndinya-Achola
J.
,
Bwayo
J.
,
Onyango
F. E.
,
Hughes
J.
,
Kreiss
J.
.
2000
.
Effect of breastfeeding and formula feeding on transmission of HIV-1: a randomized clinical trial.
JAMA
283
:
1167
1174
.
2
Taha
T. E.
,
Kumwenda
J.
,
Cole
S. R.
,
Hoover
D. R.
,
Kafulafula
G.
,
Fowler
M. G.
,
Thigpen
M. C.
,
Li
Q.
,
Kumwenda
N. I.
,
Mofenson
L.
.
2009
.
Postnatal HIV-1 transmission after cessation of infant extended antiretroviral prophylaxis and effect of maternal highly active antiretroviral therapy.
J. Infect. Dis.
200
:
1490
1497
.
3
Chasela
C. S.
,
Hudgens
M. G.
,
Jamieson
D. J.
,
Kayira
D.
,
Hosseinipour
M. C.
,
Kourtis
A. P.
,
Martinson
F.
,
Tegha
G.
,
Knight
R. J.
,
Ahmed
Y. I.
, et al
BAN Study Group
.
2010
.
Maternal or infant antiretroviral drugs to reduce HIV-1 transmission.
N. Engl. J. Med.
362
:
2271
2281
.
4
Kumwenda
N. I.
,
Hoover
D. R.
,
Mofenson
L. M.
,
Thigpen
M. C.
,
Kafulafula
G.
,
Li
Q.
,
Mipando
L.
,
Nkanaunena
K.
,
Mebrahtu
T.
,
Bulterys
M.
, et al
.
2008
.
Extended antiretroviral prophylaxis to reduce breast-milk HIV-1 transmission.
N. Engl. J. Med.
359
:
119
129
.
5
Thior
I.
,
Lockman
S.
,
Smeaton
L. M.
,
Shapiro
R. L.
,
Wester
C.
,
Heymann
S. J.
,
Gilbert
P. B.
,
Stevens
L.
,
Peter
T.
,
Kim
S.
, et al
Mashi Study Team
.
2006
.
Breastfeeding plus infant zidovudine prophylaxis for 6 months vs formula feeding plus infant zidovudine for 1 month to reduce mother-to-child HIV transmission in Botswana: a randomized trial: the Mashi Study.
JAMA
296
:
794
805
.
6
Shapiro
R. L.
,
Hughes
M. D.
,
Ogwu
A.
,
Kitch
D.
,
Lockman
S.
,
Moffat
C.
,
Makhema
J.
,
Moyo
S.
,
Thior
I.
,
McIntosh
K.
, et al
.
2010
.
Antiretroviral regimens in pregnancy and breast-feeding in Botswana.
N. Engl. J. Med.
362
:
2282
2294
.
7
John
G. C.
,
Richardson
B. A.
,
Nduati
R. W.
,
Mbori-Ngacha
D.
,
Kreiss
J. K.
.
2001
.
Timing of breast milk HIV-1 transmission: a meta-analysis.
East Afr. Med. J.
78
:
75
79
.
8
Semba
R. D.
,
Kumwenda
N.
,
Hoover
D. R.
,
Taha
T. E.
,
Quinn
T. C.
,
Mtimavalye
L.
,
Biggar
R. J.
,
Broadhead
R.
,
Miotti
P. G.
,
Sokoll
L. J.
, et al
.
1999
.
Human immunodeficiency virus load in breast milk, mastitis, and mother-to-child transmission of human immunodeficiency virus type 1.
J. Infect. Dis.
180
:
93
98
.
9
Richardson
B. A.
,
John-Stewart
G. C.
,
Hughes
J. P.
,
Nduati
R.
,
Mbori-Ngacha
D.
,
Overbaugh
J.
,
Kreiss
J. K.
.
2003
.
Breast-milk infectivity in human immunodeficiency virus type 1-infected mothers.
J. Infect. Dis.
187
:
736
740
.
10
Pillay
K.
,
Coutsoudis
A.
,
York
D.
,
Kuhn
L.
,
Coovadia
H. M.
.
2000
.
Cell-free virus in breast milk of HIV-1-seropositive women.
J. Acquir. Immune Defic. Syndr.
24
:
330
336
.
11
Rousseau
C. M.
,
Nduati
R. W.
,
Richardson
B. A.
,
Steele
M. S.
,
John-Stewart
G. C.
,
Mbori-Ngacha
D. A.
,
Kreiss
J. K.
,
Overbaugh
J.
.
2003
.
Longitudinal analysis of human immunodeficiency virus type 1 RNA in breast milk and of its relationship to infant infection and maternal disease.
J. Infect. Dis.
187
:
741
747
.
12
Rousseau
C. M.
,
Nduati
R. W.
,
Richardson
B. A.
,
John-Stewart
G. C.
,
Mbori-Ngacha
D. A.
,
Kreiss
J. K.
,
Overbaugh
J.
.
2004
.
Association of levels of HIV-1-infected breast milk cells and risk of mother-to-child transmission.
J. Infect. Dis.
190
:
1880
1888
.
13
John
G. C.
,
Nduati
R. W.
,
Mbori-Ngacha
D. A.
,
Richardson
B. A.
,
Panteleeff
D.
,
Mwatha
A.
,
Overbaugh
J.
,
Bwayo
J.
,
Ndinya-Achola
J. O.
,
Kreiss
J. K.
.
2001
.
Correlates of mother-to-child human immunodeficiency virus type 1 (HIV-1) transmission: association with maternal plasma HIV-1 RNA load, genital HIV-1 DNA shedding, and breast infections.
J. Infect. Dis.
183
:
206
212
.
14
Embree
J. E.
,
Njenga
S.
,
Datta
P.
,
Nagelkerke
N. J.
,
Ndinya-Achola
J. O.
,
Mohammed
Z.
,
Ramdahin
S.
,
Bwayo
J. J.
,
Plummer
F. A.
.
2000
.
Risk factors for postnatal mother-child transmission of HIV-1.
AIDS
14
:
2535
2541
.
15
Schmitz
J. E.
,
Kuroda
M. J.
,
Santra
S.
,
Sasseville
V. G.
,
Simon
M. A.
,
Lifton
M. A.
,
Racz
P.
,
Tenner-Racz
K.
,
Dalesandro
M.
,
Scallon
B. J.
, et al
.
1999
.
Control of viremia in simian immunodeficiency virus infection by CD8+ lymphocytes.
Science
283
:
857
860
.
16
Sabbaj
S.
,
Edwards
B. H.
,
Ghosh
M. K.
,
Semrau
K.
,
Cheelo
S.
,
Thea
D. M.
,
Kuhn
L.
,
Ritter
G. D.
,
Mulligan
M. J.
,
Goepfert
P. A.
,
Aldrovandi
G. M.
.
2002
.
Human immunodeficiency virus-specific CD8(+) T cells in human breast milk.
J. Virol.
76
:
7365
7373
.
17
Permar
S. R.
,
Kang
H. H.
,
Carville
A.
,
Mansfield
K. G.
,
Gelman
R. S.
,
Rao
S. S.
,
Whitney
J. B.
,
Letvin
N. L.
.
2008
.
Potent simian immunodeficiency virus-specific cellular immune responses in the breast milk of simian immunodeficiency virus-infected, lactating rhesus monkeys.
J. Immunol.
181
:
3643
3650
.
18
Permar
S. R.
,
Kang
H. H.
,
Wilks
A. B.
,
Mach
L. V.
,
Carville
A.
,
Mansfield
K. G.
,
Learn
G. H.
,
Hahn
B. H.
,
Letvin
N. L.
.
2010
.
Local replication of simian immunodeficiency virus in the breast milk compartment of chronically-infected, lactating rhesus monkeys.
Retrovirology
7
:
7
.
19
Heath
L.
,
Conway
S.
,
Jones
L.
,
Semrau
K.
,
Nakamura
K.
,
Walter
J.
,
Decker
W. D.
,
Hong
J.
,
Chen
T.
,
Heil
M.
, et al
.
2010
.
Restriction of HIV-1 genotypes in breast milk does not account for the population transmission genetic bottleneck that occurs following transmission.
PLoS ONE
5
:
e10213
.
20
Gantt
S.
,
Carlsson
J.
,
Heath
L.
,
Bull
M. E.
,
Shetty
A. K.
,
Mutsvangwa
J.
,
Musingwini
G.
,
Woelk
G.
,
Zijenah
L. S.
,
Katzenstein
D. A.
, et al
.
2010
.
Genetic analyses of HIV-1 env sequences demonstrate limited compartmentalization in breast milk and suggest viral replication within the breast that increases with mastitis.
J. Virol.
84
:
10812
10819
.
21
Baba
T. W.
,
Liska
V.
,
Hofmann-Lehmann
R.
,
Vlasak
J.
,
Xu
W.
,
Ayehunie
S.
,
Cavacini
L. A.
,
Posner
M. R.
,
Katinger
H.
,
Stiegler
G.
, et al
.
2000
.
Human neutralizing monoclonal antibodies of the IgG1 subtype protect against mucosal simian-human immunodeficiency virus infection.
Nat. Med.
6
:
200
206
.
22
Santra
S.
,
Barouch
D. H.
,
Korioth-Schmitz
B.
,
Lord
C. I.
,
Krivulka
G. R.
,
Yu
F.
,
Beddall
M. H.
,
Gorgone
D. A.
,
Lifton
M. A.
,
Miura
A.
, et al
.
2004
.
Recombinant poxvirus boosting of DNA-primed rhesus monkeys augments peak but not memory T lymphocyte responses.
Proc. Natl. Acad. Sci. USA
101
:
11088
11093
.
23
Harari
A.
,
Bart
P. A.
,
Stöhr
W.
,
Tapia
G.
,
Garcia
M.
,
Medjitna-Rais
E.
,
Burnet
S.
,
Cellerai
C.
,
Erlwein
O.
,
Barber
T.
, et al
.
2008
.
An HIV-1 clade C DNA prime, NYVAC boost vaccine regimen induces reliable, polyfunctional, and long-lasting T cell responses.
J. Exp. Med.
205
:
63
77
.
24
Santra
S.
,
Seaman
M. S.
,
Xu
L.
,
Barouch
D. H.
,
Lord
C. I.
,
Lifton
M. A.
,
Gorgone
D. A.
,
Beaudry
K. R.
,
Svehla
K.
,
Welcher
B.
, et al
.
2005
.
Replication-defective adenovirus serotype 5 vectors elicit durable cellular and humoral immune responses in nonhuman primates.
J. Virol.
79
:
6516
6522
.
25
Liu
J.
,
O’Brien
K. L.
,
Lynch
D. M.
,
Simmons
N. L.
,
La Porte
A.
,
Riggs
A. M.
,
Abbink
P.
,
Coffey
R. T.
,
Grandpre
L. E.
,
Seaman
M. S.
, et al
.
2009
.
Immune control of an SIV challenge by a T-cell-based vaccine in rhesus monkeys.
Nature
457
:
87
91
.
26
Liu
J.
,
Ewald
B. A.
,
Lynch
D. M.
,
Denholtz
M.
,
Abbink
P.
,
Lemckert
A. A.
,
Carville
A.
,
Mansfield
K. G.
,
Havenga
M. J.
,
Goudsmit
J.
,
Barouch
D. H.
.
2008
.
Magnitude and phenotype of cellular immune responses elicited by recombinant adenovirus vectors and heterologous prime-boost regimens in rhesus monkeys.
J. Virol.
82
:
4844
4852
.
27
Mascola
J. R.
,
Sambor
A.
,
Beaudry
K.
,
Santra
S.
,
Welcher
B.
,
Louder
M. K.
,
Vancott
T. C.
,
Huang
Y.
,
Chakrabarti
B. K.
,
Kong
W. P.
, et al
.
2005
.
Neutralizing antibodies elicited by immunization of monkeys with DNA plasmids and recombinant adenoviral vectors expressing human immunodeficiency virus type 1 proteins.
J. Virol.
79
:
771
779
.
28
Baig
J.
,
Levy
D. B.
,
McKay
P. F.
,
Schmitz
J. E.
,
Santra
S.
,
Subbramanian
R. A.
,
Kuroda
M. J.
,
Lifton
M. A.
,
Gorgone
D. A.
,
Wyatt
L. S.
, et al
.
2002
.
Elicitation of simian immunodeficiency virus-specific cytotoxic T lymphocytes in mucosal compartments of rhesus monkeys by systemic vaccination.
J. Virol.
76
:
11484
11490
.
29
Kaufman
D. R.
,
Liu
J.
,
Carville
A.
,
Mansfield
K. G.
,
Havenga
M. J.
,
Goudsmit
J.
,
Barouch
D. H.
.
2008
.
Trafficking of antigen-specific CD8+ T lymphocytes to mucosal surfaces following intramuscular vaccination.
J. Immunol.
181
:
4188
4198
.
30
Munoz
F. M.
,
Piedra
P. A.
,
Glezen
W. P.
.
2003
.
Safety and immunogenicity of respiratory syncytial virus purified fusion protein-2 vaccine in pregnant women.
Vaccine
21
:
3465
3467
.
31
Shahid
N. S.
,
Steinhoff
M. C.
,
Hoque
S. S.
,
Begum
T.
,
Thompson
C.
,
Siber
G. R.
.
1995
.
Serum, breast milk, and infant antibody after maternal immunisation with pneumococcal vaccine.
Lancet
346
:
1252
1257
.
32
Finn
A.
,
Zhang
Q.
,
Seymour
L.
,
Fasching
C.
,
Pettitt
E.
,
Janoff
E. N.
.
2002
.
Induction of functional secretory IgA responses in breast milk, by pneumococcal capsular polysaccharides.
J. Infect. Dis.
186
:
1422
1429
.
33
Deubzer
H. E.
,
Obaro
S. K.
,
Newman
V. O.
,
Adegbola
R. A.
,
Greenwood
B. M.
,
Henderson
D. C.
.
2004
.
Colostrum obtained from women vaccinated with pneumococcal vaccine during pregnancy inhibits epithelial adhesion of Streptococcus pneumoniae.
J. Infect. Dis.
190
:
1758
1761
.
34
Hahn-Zoric
M.
,
Carlsson
B.
,
Jalil
F.
,
Mellander
L.
,
Germanier
R.
,
Hanson
L. A.
.
1989
.
The influence on the secretory IgA antibody levels in lactating women of oral typhoid and parenteral cholera vaccines given alone or in combination.
Scand. J. Infect. Dis.
21
:
421
426
.
35
Merson
M. H.
,
Black
R. E.
,
Sack
D. A.
,
Svennerholm
A. M.
,
Holmgren
J.
.
1980
.
Maternal cholera immunisation and scecretory IgA in breast milk.
Lancet
1
:
931
932
.
36
Mascart-Lemone
F.
,
Carlsson
B.
,
Jalil
F.
,
Hahn-Zoric
M.
,
Duchateau
J.
,
Hanson
L. A.
.
1988
.
Polymeric and monomeric IgA response in serum and milk after parenteral cholera and oral typhoid vaccination.
Scand. J. Immunol.
28
:
443
448
.
37
Losonsky
G. A.
,
Fishaut
J. M.
,
Strussenberg
J.
,
Ogra
P. L.
.
1982
.
Effect of immunization against rubella on lactation products. I. Development and characterization of specific immunologic reactivity in breast milk.
J. Infect. Dis.
145
:
654
660
.
38
Chakrabarti
B. K.
,
Kong
W. P.
,
Wu
B. Y.
,
Yang
Z. Y.
,
Friborg
J.
,
Ling
X.
,
King
S. R.
,
Montefiori
D. C.
,
Nabel
G. J.
.
2002
.
Modifications of the human immunodeficiency virus envelope glycoprotein enhance immunogenicity for genetic immunization.
J. Virol.
76
:
5357
5368
.
39
Huang
Y.
,
Kong
W. P.
,
Nabel
G. J.
.
2001
.
Human immunodeficiency virus type 1-specific immunity after genetic immunization is enhanced by modification of Gag and Pol expression.
J. Virol.
75
:
4947
4951
.
40
Abbink
P.
,
Lemckert
A. A.
,
Ewald
B. A.
,
Lynch
D. M.
,
Denholtz
M.
,
Smits
S.
,
Holterman
L.
,
Damen
I.
,
Vogels
R.
,
Thorner
A. R.
, et al
.
2007
.
Comparative seroprevalence and immunogenicity of six rare serotype recombinant adenovirus vaccine vectors from subgroups B and D.
J. Virol.
81
:
4654
4663
.
41
Sun
Y.
,
Bailer
R. T.
,
Rao
S. S.
,
Mascola
J. R.
,
Nabel
G. J.
,
Koup
R. A.
,
Letvin
N. L.
.
2009
.
Systemic and mucosal T-lymphocyte activation induced by recombinant adenovirus vaccines in rhesus monkeys.
J. Virol.
83
:
10596
10604
.
42
Sun
Y.
,
Schmitz
J. E.
,
Buzby
A. P.
,
Barker
B. R.
,
Rao
S. S.
,
Xu
L.
,
Yang
Z. Y.
,
Mascola
J. R.
,
Nabel
G. J.
,
Letvin
N. L.
.
2006
.
Virus-specific cellular immune correlates of survival in vaccinated monkeys after simian immunodeficiency virus challenge.
J. Virol.
80
:
10950
10956
.
43
Li
M.
,
Gao
F.
,
Mascola
J. R.
,
Stamatatos
L.
,
Polonis
V. R.
,
Koutsoukos
M.
,
Voss
G.
,
Goepfert
P.
,
Gilbert
P.
,
Greene
K. M.
, et al
.
2005
.
Human immunodeficiency virus type 1 env clones from acute and early subtype B infections for standardized assessments of vaccine-elicited neutralizing antibodies.
J. Virol.
79
:
10108
10125
.
44
Sun
Y.
,
Santra
S.
,
Schmitz
J. E.
,
Roederer
M.
,
Letvin
N. L.
.
2008
.
Magnitude and quality of vaccine-elicited T-cell responses in the control of immunodeficiency virus replication in rhesus monkeys.
J. Virol.
82
:
8812
8819
.
45
McElrath
M. J.
,
De Rosa
S. C.
,
Moodie
Z.
,
Dubey
S.
,
Kierstead
L.
,
Janes
H.
,
Defawe
O. D.
,
Carter
D. K.
,
Hural
J.
,
Akondy
R.
, et al
Step Study Protocol Team
.
2008
.
HIV-1 vaccine-induced immunity in the test-of-concept Step Study: a case-cohort analysis.
Lancet
372
:
1894
1905
.
46
Buchbinder
S. P.
,
Mehrotra
D. V.
,
Duerr
A.
,
Fitzgerald
D. W.
,
Mogg
R.
,
Li
D.
,
Gilbert
P. B.
,
Lama
J. R.
,
Marmor
M.
,
Del Rio
C.
, et al
Step Study Protocol Team
.
2008
.
Efficacy assessment of a cell-mediated immunity HIV-1 vaccine (the Step Study): a double-blind, randomised, placebo-controlled, test-of-concept trial.
Lancet
372
:
1881
1893
.
47
Rychert
J.
,
Amedee
A. M.
.
2005
.
The antibody response to SIV in lactating rhesus macaques.
J. Acquir. Immune Defic. Syndr.
38
:
135
141
.
48
Van de Perre
P.
,
Simonon
A.
,
Hitimana
D. G.
,
Dabis
F.
,
Msellati
P.
,
Mukamabano
B.
,
Butera
J. B.
,
Van Goethem
C.
,
Karita
E.
,
Lepage
P.
.
1993
.
Infective and anti-infective properties of breastmilk from HIV-1-infected women.
Lancet
341
:
914
918
.
49
Kuhn
L.
,
Trabattoni
D.
,
Kankasa
C.
,
Sinkala
M.
,
Lissoni
F.
,
Ghosh
M.
,
Aldrovandi
G.
,
Thea
D.
,
Clerici
M.
.
2006
.
Hiv-specific secretory IgA in breast milk of HIV-positive mothers is not associated with protection against HIV transmission among breast-fed infants.
J. Pediatr.
149
:
611
616
.
50
Rerks-Ngarm
S.
,
Pitisuttithum
P.
,
Nitayaphan
S.
,
Kaewkungwal
J.
,
Chiu
J.
,
Paris
R.
,
Premsri
N.
,
Namwat
C.
,
de Souza
M.
,
Adams
E.
, et al
MOPH-TAVEG Investigators
.
2009
.
Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand.
N. Engl. J. Med.
361
:
2209
2220
.
51
Imaoka
K.
,
Miller
C. J.
,
Kubota
M.
,
McChesney
M. B.
,
Lohman
B.
,
Yamamoto
M.
,
Fujihashi
K.
,
Someya
K.
,
Honda
M.
,
McGhee
J. R.
,
Kiyono
H.
.
1998
.
Nasal immunization of nonhuman primates with simian immunodeficiency virus p55gag and cholera toxin adjuvant induces Th1/Th2 help for virus-specific immune responses in reproductive tissues.
J. Immunol.
161
:
5952
5958
.
52
Pinczewski
J.
,
Zhao
J.
,
Malkevitch
N.
,
Patterson
L. J.
,
Aldrich
K.
,
Alvord
W. G.
,
Robert-Guroff
M.
.
2005
.
Enhanced immunity and protective efficacy against SIVmac251 intrarectal challenge following ad-SIV priming by multiple mucosal routes and gp120 boosting in MPL-SE.
Viral Immunol.
18
:
236
243
.
53
Wang
S. W.
,
Bertley
F. M.
,
Kozlowski
P. A.
,
Herrmann
L.
,
Manson
K.
,
Mazzara
G.
,
Piatak
M.
,
Johnson
R. P.
,
Carville
A.
,
Mansfield
K.
,
Aldovini
A.
.
2004
.
An SHIV DNA/MVA rectal vaccination in macaques provides systemic and mucosal virus-specific responses and protection against AIDS.
AIDS Res. Hum. Retroviruses
20
:
846
859
.
54
Sabbaj
S.
,
Ghosh
M. K.
,
Edwards
B. H.
,
Leeth
R.
,
Decker
W. D.
,
Goepfert
P. A.
,
Aldrovandi
G. M.
.
2005
.
Breast milk-derived antigen-specific CD8+ T cells: an extralymphoid effector memory cell population in humans.
J. Immunol.
174
:
2951
2956
.
55
Amedee
A. M.
,
Lacour
N.
,
Ratterree
M.
.
2003
.
Mother-to-infant transmission of SIV via breast-feeding in rhesus macaques.
J. Med. Primatol.
32
:
187
193
.