of March 8, 2012This information is current as

http://www.jimmunol.org/content/185/11/7097doi:10.4049/jimmunol.1002751November 2010;

2010;185;7097-7106; Prepublished online 1J Immunol Letvin and Sallie R. PermarEsteban, Giuseppe Pantaleo, Dan H. Barouch, Norman L.Piya Sircar, Angela Carville, Carmen E. Gomez, Mariano Andrew B. Wilks, Elizabeth C. Christian, Michael S. Seaman, Rhesus MonkeysLive Virus Vector Boost Vaccination of Lactating Simian Immunodeficiency Virus DNA Prime andResponses in Breast Milk following Systemic Robust Vaccine-Elicited Cellular Immune

References

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The Journal of Immunology

Robust Vaccine-Elicited Cellular Immune Responses inBreast Milk following Systemic Simian ImmunodeficiencyVirus DNA Prime and Live Virus Vector Boost Vaccination ofLactating Rhesus Monkeys

Andrew B. Wilks,* Elizabeth C. Christian,* Michael S. Seaman,*,† Piya Sircar,*

Angela Carville,‡ Carmen E. Gomez,x Mariano Esteban,x Giuseppe Pantaleo,{,‖

Dan H. Barouch,† Norman L. Letvin,* and Sallie R. Permar*,#

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 Ap01+ 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. The Journal of Immunology, 2010, 185: 7097–7106.

HIV transmission via breast milk accounts for nearly halfof the 350,000 new infant infections occurring annuallyin developing regions with high HIV prevalence (1).

Although infant or maternal antiretroviral prophylaxis can reducethe incidence of breast milk HIV transmission (2–6), effectiveimplementation of these long-term prophylaxis regimens will becomplicated by infant toxicity, compliance, and inadequate health

care infrastructure. Therefore, investigations of immunologic inter-ventions, such as maternal or infant vaccines, to reduce postnataltransmission of HIV remain crucial for elimination of this modeof HIV transmission. Interestingly, in the absence of antiretro-viral prophylaxis, only 10% of breastfeeding infants born to HIV-infected mothers will become infected postnatally (7). This lowrate of HIV acquisition, despite daily low-dose exposure for upto 2 y, raises the possibility that protective immune responsesin breast milk may prevent HIV acquisition in the majority ofbreastfeeding infants.The risk for HIV transmission via breastfeeding has been asso-

ciated with the level of HIV RNA and cell-associated HIV DNA inmilk (8–12), in addition to maternal disease progression (8, 13, 14).Therefore, the reduction of cell-free and cell-associated virus loadthrough enhancement of virus-specific immune responses in milkmay result in reduced rates of HIV transmission via breast milk.HIV/SIV-specific CD8+ T lymphocyte responses are known to becritical for containment of systemic virus load (15) and have beenidentified in human and rhesus monkey milk (16, 17). Furthermore,recent studies of HIV/SIV virus evolution in milk suggest that thevirus may replicate locally in the breast milk compartment (18–20). Therefore, enhancement of the local HIV-specific CD8+ Tlymphocyte responses in breast milk may improve containment ofvirus replication in the breast milk compartment and result ina decrease in breast milk virus load. In addition, passive postnataladministration of broadly HIV-neutralizing Ig protected infantmacaques from HIV acquisition by oral exposure (21). Therefore,induction of potent, functional HIV-specific Ab responses in breast

*Division of Viral Pathogenesis, †Division of Vaccine Research, Beth Israel Deacon-ess Medical Center, and #Division of Infectious Disease, Children’s Hospital Boston,Harvard Medical School, Boston, MA 02115; ‡New England Regional Primate Re-search Center, Harvard Medical School, Southboro, MA 01772; xDepartamento deBiologıa Celular y Molecular, Centro Nacional de Biotecnologia, Consejo Superiorde Investigaciones Cientıficas, Ciudad Universitaria Cantoblanco, Madrid, Spain;{Laboratory of AIDS Immunopathogenesis, Division of Immunology and Allergy,Department of Medicine, Central University Hospital of Vaudois, University of Lau-sanne; and ‖Swiss Vaccine Research Institute, Lausanne, Switzerland

Received for publication August 13, 2010. Accepted for publication September 28,2010.

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

Address correspondence and reprint requests to Dr. Sallie R. Permar, 330 BrooklineAvenue, E/CLS 1042 Beth Israel Deaconess Medical Center, Boston, MA 02115.E-mail address: sallie.permar@childrens.harvard.edu

Abbreviations used in this paper: rAd5, replication-incompetent adenovirus vectorserotype 5; SEB, Staphylococcus endotoxin B; TCLA, T cell line adapted.

Copyright� 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00

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milk through maternal vaccination may play a role in blocking HIVacquisition 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 oflactating monkeys. Nonhuman primates have proved to be an in-strumental model for evaluating immunogenicity and efficacy ofcandidate HIV vaccines. Furthermore, we established a protocol forhormone-induction of lactation to investigate cellular and humoralimmune responses in breast milk, because the breast milk producedby the hormone induction is immunologically similar to that ofnaturally lactating monkeys (17). It has long been noted that rDNApriming prior to virus vector boost enhances vaccine-elicited cel-lular immune responses (22); therefore, the monkeys in this studywere primed with rDNA containing SIV gag, pol, and envelopeimmunogens. The monkeys were then boosted with matchingSIV immunogens delivered via the attenuated poxvirus vector,NYVAC, a vector that induces strong polyfunctional CD4+ andCD8+ T lymphocyte responses (23), and replication-incompetentadenovirus vector serotype 5 (rAd5), a vector that induces potentcellular and humoral immune responses (24–27). Adenovirus andpoxvirus vectors were reported to induce mucosal CD8+ T lym-phocyte responses in the gastrointestinal tract (28, 29), makingthese vectors excellent candidates for eliciting immune responsesin breast milk.Although induction of humoral immune responses in milk fol-

lowing systemic vaccination with polysaccharide, protein, or whole-cell vaccines and elicitation of cellular immune responses in milkfollowing live-attenuated virus vaccination were reported (30–37),it has not been established whether any candidate HIV vaccine canelicit humoral and/or cellular immune responses in breast milk.Therefore, our study aimed to characterize systemic cellular andhumoral immune responses in breast milk following SIV vacci-nation. This detailed characterization of cellular and humoral im-mune responses in milk following systemic vaccination has impli-cations for mother-to-child transmission of HIV and other neonatalpathogens transmitted via breast milk, such as human T lympho-trophic virus-1 and CMV. This study of breast milk immune re-sponses elicited by a leading candidate HIV vaccine strategy willform the basis of future investigations of maternal vaccination toinduce virus-specific immune responses that are protective againstvirus transmission via breastfeeding.

Materials and MethodsGeneration of vaccine vectors

DNA plasmids containing the SIVmac239 gag-pol, and envelope genes,were generated by cloning the virus cDNA into the pVR1012 vector, aspreviously described (38, 39). Replication-incompetent, E1/E3-deletedrAd5 vectors expressing the SIVmac239 Gag and Envelope and SIVE660 Pol were prepared, as previously described (40). Finally, SIVmac239gene-expressing NYVAC vectors were generated by the following pro-tocol: plasmid transfer vectors pCyA20-SIVgag-pol and gp140 wereconstructed for insertion of the SIVmac239 gag-pol and envelope genesinto the TK locus of the NYVAC genome. After the desired recombinantplasmids were isolated by screening for expression of b-galactosidaseactivity, BSC40 cells were infected with NYVAC virus (provided bySanofi-Aventis, Paris, France) at a multiplicity of 0.05 PFU/cell andtransfected with 10 mg DNA of the plasmid transfer vector using Lip-ofectamine reagent (Invitrogen, Carlsbad, CA). After 72 h, cells wereharvested, sonicated, and used for recombinant virus screening. RecobinantNYVAC viruses containing the SIV genes and transiently coexpressing theb-gal marker gene were selected by four consecutive rounds of plaquepurification in BSC40 cells stained with 5-bromo-4-chloro-3-indolylb-galactoside (300 mg/ml). Next, recombinant NYVAC viruses containingthe SIV genes and having the b-gal marker gene deleted through re-combination at the TK site were isolated by three additional consecutiverounds of plaque purification, screening for nonstaining viral foci in BSC40

cells. Isolated plaques were grown and analyzed for the presence and ex-pression of SIV gene products and for the absence of NYVAC-WT con-tamination by PCR and Western blot with SIV protein-specific mAbs(provided by Project EVA, European Union). Virus stocks were then gen-erated in CEF cells and purified by two 45% sucrose gradient purifications.

Animals and vaccination

Lactation was pharmacologically induced in five female Mamu-Ap01+

rhesus monkeys using depot medroxyprogesterone and estradiol injectionsand an oral dopamine antagonist, as previously described (17). Once allfive monkeys were lactating, the animals were vaccinated i.m. with 5 mgeach rDNA plasmid containing the SIVmac239 gag-pol and envelopegenes on weeks 0, 4, and 8. The monkeys were boosted i.m. and in-tradermally at week 28 with 108 PFU each of the NYVAC vectors con-taining the SIVmac239 gag-pol and envelope genes. The monkeys wereboosted again at week 38 by i.m. administration of 1010 viral particles ofeach of the rAd5 vectors containing the SIVmac239 gag and envelopegenes and SIV E660 pol gene. Milk (30–1000 ml) was collected two timesper week throughout the vaccination schedule, and blood was collectedweekly. Milk was separated into cellular, supernatant, and fat fractions bycentrifugation, as previously described (17). Pinch biopsies from the colonand vaginal tract obtained at week 40 were digested with collagenase, andmononuclear cells were isolated on a Percoll gradient within 6 h, as pre-viously described (41). Animals were maintained according to the “Guidefor the Care and Use of Laboratory Animals.”

Phenotyping, tetramer staining, and intracellular cytokinestaining of mucosal biopsies, breast milk cells, and PBMCs

The vaccine-elicited CD8+ T lymphocyte response was measured bystaining milk, colon, and vaginal cells and PBMCs with immunodominantMamu Ap01-restricted Env p54-allophycocyanin and Gag p11C-Q-Dot655-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 BDBiosciences, San Jose, CA) mAb staining. Naive and memory lymphocytesubsets were defined by anti–CD95-allophycocyanin (DX2; BD Bio-sciences) and anti–CD28-PerCP-Cy5.5 (28.2; Beckman Coulter, Fullerton,CA) staining (naive: CD28+, CD952; effector memory: CD282, CD95+;central memory: CD28+, CD95+). The peak response was defined as thehighest proportion of tetramer-staining CD8+ T lymphocytes measuredafter each vaccination (between 2 and 4 wk following each immunizationfor each monkey).

Activation status of breast milk CD4+ T lymphocytes after vaccinationwas 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), allfrom BD Biosciences]. Intracellular cytokine staining was performed aftera 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-a–Pacific Blue (Mab11;eBiosciences, San Diego, CA), anti–IFN-g–PE-Cy7 (B27; BD Biosci-ences), and anti–IL-2–allophycocyanin (MQ1-17H12; BD Biosciences). Theproportion of cytokine-producing cells was determined by subtracting theproportion of unstimulated cytokine-producing cells from the proportion ofGag-stimulated cytokine-producing cells. An amine dye (Aqua Amine) wasused to distinguish live from dead cells in all flow-cytometric analyses. Datawere collected on the LSRII flow cytometer (BD Biosciences) with FACS-diva software and analyzed with FlowJo Software.

Absolute lymphocyte and SIV-specific CD8+ T lymphocytecount

Automated complete cell counts of breast milk are skewed by the presenceof fat droplets; therefore, they cannot be used to quantitate lymphocytenumber. To calculate absolute lymphocytes per milliliter of milk and bloodin a similar fashion, the absolute number of CD45+CD3+ lymphocytesquantitated by flow-cytometric analysis of each sample type was normal-ized to the ratio of fluorospheres added to the sample and quantitated byflow cytometry at the end of each staining procedure. To calculate theabsolute lymphocyte count in blood, 100 ml EDTA-anticoagulated bloodwas mixed with 100 ml Flow-Count Fluorospheres (Beckman Coulter; 942beads/ml) and stained with anti–CD45-FITC (DO58-1283) and anti–CD3-allophycocyanin-Cy7 (SP34.2; both from BD Biosciences). The RBCswere lysed and fixed using a TQ-Prep machine (Beckman Coulter), and theremaining blood cells and fluorospheres were washed, pelleted, and fixed.To calculate the absolute lymphocyte count in breast milk, 100 ml Flow-Count Fluorospheres were added to the total breast milk cell pellet. Breast

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milk cells were stained with the phenotyping Abs listed above. Sampledata were collected within 4 h of fixation on the LSRII instrument (BDBiosciences) with FACSDiva software and analyzed with FlowJo software.After flow-cytometric analysis and quantitation of the fluorospheres thatremained at the end of the procedure, the number of CD45+CD3+ lym-phocyte events was normalized by the ratio of the number of fluorospheresadded to the sample/the number of fluorospheres quantitated by flowcytometry. Finally, the normalized number of CD45+CD3+ lymphocyteswas divided by the volume of the original sample. Absolute CD4+, CD8+,Gag p11c-specific CD8+, and Env p45-specific CD8+ T lymphocytes permilliliter were calculated by multiplying the percentage of the populationby the normalized absolute CD45+CD3+ lymphocyte count. The peak re-sponse was defined as the highest absolute number of tetramer-stainingCD8+ T lymphocytes measured after each vaccination (between 2 and 4wk following each immunization for each monkey).

Quantitation of SIV envelope-binding IgG, IgA, andneutralizing titer in milk and plasma

SIV Envelope-binding IgG and IgAwere measured by incubation of serial3-fold dilutions of plasma and milk supernatant in duplicate in a 96-wellplate coated with rSIVmac239 gp130 (ImmunoDiagnostics, Woburn, MA).After blocking with PBS with 5% nonfat dried milk and 10% FBS, SIVEnvelope-binding Ab was detected by an HRP-conjugated, polyclonal goatanti-monkey IgG (Alpha Diagnostics, Owings Mills, MD) or anti-monkeyIgA (Rockland, Gilbertsville, PA) Ab and the addition of ABTS-2 per-oxidase substrate system (Kirkegaard & Perry Laboratories, Gaithersburg,MD). OD was measured at 450 nm. SIV envelope-specific Ab titer wascalculated as the inverse of the lowest dilution of plasma or milk super-natant that had an average OD that was 2-fold greater than that of the PBSnegative control.

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

Statistical analysis

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

ResultsSIV vaccination and assessment of changes in lymphocytenumber and phenotype in milk of lactating rhesus monkeys

Five Mamu Ap01+ hormone-induced lactating female rhesus mon-keys received priming immunizations i.m. with 5 mg of rDNAplasmids with SIVmac239 gag-pol and env gene inserts on weeks0, 4, and 8. Animals were boosted i.m. and intradermally at week28 with 108 PFU of recombinant attenuated pox vector, NYVAC,containing SIVmac239 gag-pol and env genes. Finally, animalswere repeat boosted i.m. at week 38 with 1010 viral particles ofrecombinant adenovirus serotype 5 containing SIV gag, pol, andenvelope. The vaccine-elicited cellular and humoral immune re-sponses were assessed in blood and breast milk following eachimmunization. Animals underwent colon and vaginal biopsy 2 wkfollowing the final immunization to compare the magnitude of the

cellular immune responses in breast milk with these mucosal com-partments (Fig. 1).We first investigated the changes in lymphocyte number and

relative proportions of CD4+ and CD8+ T memory and naivelymphocytes in milk and blood weekly following systemic vac-cination. The lymphocyte number and the proportion of CD8+

effector memory T lymphocytes increased greatly following SIVinfection (17), but changes in lymphocyte number and phenotypein milk following maternal vaccination have not been studied. Theabsolute number of lymphocytes in breast milk before vaccination(median, 4300 CD3+ T cells/ml; range: 1,400–41,800) did nottrend toward a statistically significant difference at the peak of theresponse after each immunization (data not shown). Furthermore,the proportion of CD4+ (median, 64.3%; range: 30–72.4%) orCD8+ (median, 32.5%; range: 25.8–72.8%) T lymphocytes in milkprior to vaccination did not change following each systemic im-munization (data not shown). Finally, the proportion of effectormemory (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+ Tlymphocytes did not significantly change in milk following eachsystemic immunization (data not shown). Accordingly, there wereno changes in the number and proportion of memory and naiveCD4+ and CD8+ T lymphocytes in blood following vaccination(data not shown).

Robust CD8+ T lymphocyte responses in breast milk ofSIV-vaccinated, lactating rhesus monkeys

The vaccine-elicited CD8+ T lymphocyte response specific for theimmunodominant Mamu Ap01-restricted epitope, Gag p11C, andthe subdominant Mamu Ap01-restricted epitope, Env p54, wereassessed weekly following each immunization. The peak pro-portion of CD8+ T lymphocytes that were specific for the Gagp11C 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,

FIGURE 1. Study design and vaccine regimen in lactating rhesus mon-

keys. 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 per-

formed 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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the peak proportion of Gag p11C-specific CD8+ T lymphocytes inbreast milk (median, 1.6%; range: 1.3–3.7%) was similar inmagnitude 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 Gagp11C-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 com-partment were not different enough to approach statistical sig-nificance (p = 0.31) (Fig. 2A, 2B). Despite the higher-magnitudeGag p11C-specific CD8+ T lymphocyte response in breast milk,the median absolute number of Gag p11C-specific CD8+ T lym-phocytes remained one to two logs lower in milk than in the bloodthroughout the immunization schedule as a result of the lowlymphocyte number in breast milk (p = 0.06 for all comparisons ofblood and milk absolute Gag p11C-specific CD8+ T lymphocytenumber after each immunization) (Fig. 2C, 2D).The vaccine-elicited CD8+ T lymphocyte response specific for

the subdominant Env p54 epitope was of greater magnitude in milkthan 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 tothe vaccine-elicited immunodominant Gag p11C-specific CD8+

T lymphocyte number in milk, the median absolute number ofvaccine-elicited Env p54-specific CD8+ T lymphocytes in milk wassimilar 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 absolutenumber of vaccine-elicited Env p54-specific CD8+ T lymphocytesdid trend toward being significantly lower in milk (median, 84cells/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 boostare summarized in Table I.

Strong vaccine-elicited, polyfunctional CD8+ and CD4+

T lymphocyte responses in breast milk

We were able to demonstrate SIV Gag-specific IFN-g, TNF-a, andIL-2 production in CD4+ and CD8+ T lymphocytes in breast milkby intracellular cytokine staining 3 wk following rDNA priming,as well as NYVAC and rAd5 boost in two vaccinated monkeyswith adequate lymphocyte number (CD3+ T lymphocyte number. 1000 per condition) (Fig. 3). The proportion of Gag-specificcytokine-producing CD4+ T lymphocytes in milk was similar tothat in blood following rDNA prime. However, the proportion ofGag-specific, IFN-g–producing CD4+ T lymphocytes was$5-foldhigher in milk than in blood in both animals following NYVACimmunization. In addition, the proportions of Gag-specific TNF-aand IL-2–producing CD4+ T lymphocytes were $5-fold higher inmilk than in blood in one animal and were similar or greater inmagnitude in the other animal following NYVAC. Finally, theproportion of Gag-specific IFN-g–, TNF-a–, and IL-2–producingCD4+ T lymphocytes was $4-fold higher in milk than in blood ofboth animals following rAd5 vaccination. As expected, the mag-nitude of the vaccine-elicited CD4+ T lymphocyte responses was

FIGURE 2. Robust vaccine-elicited CD8+ T

lymphocyte responses appear in breast milk fol-

lowing rDNA, pox vector, and recombinant ade-

novirus 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 immuniza-

tion. 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) fol-

lowing each immunization.

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higher in blood and milk following NYVAC boost compared withthose elicited by rAd5 boost, because poxvirus vectors are knownto elicit strong CD4+ T lymphocyte responses (44) (Fig. 4A, 4B).The proportions of Gag-specific cytokine-producing CD8+ T

lymphocytes in milk were similar or higher than that in blood afterrDNA priming and NYVAC and rAd5 boost. Of note, the pro-portions of Gag-specific TNF-a– and IL-2–producing CD8+ Tlymphocytes in milk were $1 log higher than that in blood in bothmonkeys following rAd5 boost. Furthermore, the proportion ofGag-specific IFN-g–producing CD8+ T lymphocytes was 1-loghigher in milk of one animal than that in blood following rAd5boost. As expected, the proportions of Gag-specific TNF-a– andIL-2–producing CD8+ T lymphocytes in milk were higher in bothmonkeys following rAd5 boost than those following rDNA primeand NYVAC boost, because rAd5 vectors are known to inducestrong 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 vaccinevector boost. Interestingly, a distinct polyfunctional cytokine-production profile of the vaccine-elicited T lymphocyte was eli-cited in milk compared with that in blood (Fig. 4E–H). AlthoughGag-specific IFN-g only-producing cells were a predominant pop-ulation in blood and milk CD4+ and CD8+ T lymphocyte subsets,the TNF-a– and IFN-g–producing cells were predominant onlyin milk vaccine-elicited CD4+ and CD8+ T lymphocytes. Fur-thermore, the TNF only-producing cells and IL-2, TNF-, and IFN-producing populations accounted for a higher proportion of cy-tokine-producing cells in milk CD4+ and CD8+ T lymphocytesthan in the blood (Fig. 4E–H). This same pattern was found afterNYVAC and rAd5 vector boost. This difference in the polyfunc-tional cytokine profile of vaccine-elicited T lymphocytes in milkand blood likely reflects the regulated trafficking of activatedlymphocytes into the breast milk compartment or local proliferationof memory lymphocytes in milk.

The magnitude of the vaccine-elicited CD8+ T lymphocyteimmune responses in breast milk is greater than that in thecolon and similar to that in the vaginal tract

We compared the proportion of Gag p11C-specific CD8+ T lym-phocytes in blood, milk, colon, and vaginal tract 2 wk followingthe rAd5 boost. The median proportion of vaccine-elicited CD8+ Tlymphocytes specific for the immunodominant Gag p11C epitopein milk (median, 5.4%; range: 2.5–9.1%) was higher than that incolon (median, 2.2%; range: 0.7–2.9%) and trended toward a sta-tistically significantly different (p = 0.12). In contrast, the medianproportion of vaccine-elicited CD8+ T lymphocytes specific forGag 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 magnitudecompared with that elicited in other mucosal compartments.

Low-magnitude SIV envelope-specific IgG responses areelicited in breast milk by systemic rDNA prime and virus vectorboost

Vaccine-elicited neutralizing Ab responses were detected in milkagainst a TCLASIVmac239 in a pseudovirus TZM-bl–neutralizationassay after NYVAC and rAd5 boost (Fig. 6B). However, this neu-tralizing Ab response inmilk (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 IgGresponse was detected in milk after NYVAC (median titer, 3; range:3–100) and rAd5 (median titer, 10; range: 3–100) boost, but the titerwas 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 SIVEnvelope-binding IgA response was only detected in milk of oneanimal following NYVAC boost and in no animal after rAd5 boost(Fig. 6F). This limited detection of SIV Envelope-binding IgA

Table I. Median vaccine-elicited cellular and humoral immune responses in blood and milk of systemicallyvaccinated, lactating rhesus monkeys

Immune Parameter Vaccination Blood Milk

Gag p11C-specific CD8+ T cells (%) Prevaccination 0.01 0rDNA 0.4 2.9NYVAC 1.1 1.7rAd5 3.5 7.3

Abs Gag p11C-specific CD8+ T cells (cells/ml) Prevaccination 25 0rDNA 1,600 391NYVAC 10,900 93rAd5 18,700 873

Env p54-specific CD8+ T cells (%) Prevaccination 0 0rDNA 0.2 0.9NYVAC 0.24 0.7rAd5 0.1 2.3

Abs Env p54-specific CD8+ T cells (cell/ml) Prevaccination 0 0rDNA 749 119NYVAC 1,727 84rAd5 985 266

TCLA SIV-neutralizing Ab (ID50 titer) Prevaccination 13 11rDNA 1,047 27NYVAC 1,995 24rAd5 10,422 107

Anti-SIV gp130 IgG (titer) Prevaccination 0 0rDNA 0 300NYVAC 3 10,000rAd5 10 10,000

Anti-SIV gp130 IgA (titer) Prevaccination 0 0rDNA 0 0NYVAC 65 0rAd5 30 0

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response in milk is in contrast to the systemic SIV Envelope-bindingIgA response that was detected in all animals after NYVAC (mediantiter, 30; range: 30–300) and rAd5 (median titer, 30; range: 30–100)boost (Fig. 6E). Therefore, the vaccine-elicited neutralizing Ab re-

sponse in milk is likely attributable to the SIVenvelope-specific IgGin milk rather than SIVenvelope-specific secretory IgA. The medianSIV envelope-binding and -neutralizing Ab responses after eachvaccine vector are summarized in Table I.

FIGURE 4. The vaccine-elicited CD4+ and

CD8+ T lymphocyte responses in milk are of

higher magnitude and display a distinct poly-

functional 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-g, TNF-a, 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 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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Limited and transient activation of CD4+ T lymphocytes inmilk following systemic rDNA prime and live vector virus boostof uninfected monkeys

Because activation of CD4+ T lymphocytes following vaccinationhas been raised as a concern for enhancing virus transmission (45,46), we investigated the activation status of the CD4+ T lympho-cytes in milk by assessing their IL-2R (CD25), MHC class IImolecule HLA-DR, and the HIV coreceptor CCR5 expressionthroughout the immunization schedule. There was a 2–10% in-crease in the proportion of CD4+ T lymphocytes in milk expressingCCR5 following the second and third systemic rDNA prime, butthis increase lasted #2 wk. Because the median number of CD4+

T lymphocytes in milk during the vaccination schedule was 2752cells/ml, the maximum absolute increase in CCR5-expressingCD4+ T lymphocytes following DNA vaccination is 275 cells/ml.There was no apparent increase in CCR5 expression followingNYVAC boost; however, there was a 1–3% increase in the pro-portion of CCR5-expressing cells lasting #2 wk following rAd5boost (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 in-creased by 1–5% following the second and third rDNA prime, butagain this increase lasted #2 wk. This increased proportion ofCD25-expressing CD4+ T lymphocytes following DNA primingrepresents a maximum absolute increase of 137 CD25-expressingCD4+ T cells/ml. There was a minimal increase in the proportion ofCD25-expressing CD4+ T lymphocytes following NYVAC andrAd5 boost (Fig. 7B). Similarly, the proportion of CD4+ T lym-phocytes expressing HLA-DR increased 5–10% following thesecond and third rDNA prime, but this increase lasted ,2 wkand represents a maximum absolute increase of 275 HLA-DR–expressing CD4+ T lymphocytes/ml. Finally, there was a smallincrease in the proportion of CD4+ T lymphocytes expressingHLA-DR in only one of five monkeys following NYVAC boost andin two of five monkeys following rAd5 boost that lasted ,2 wk(Fig. 7C). Furthermore, there was no significant change in the meanfluorescence intensity of the CCR5-, CD25-, or HLA-DR–ex-pressing cells after vaccination to indicate a higher level ofactivation-molecule expression (data not shown). Therefore, theactivation of breast milk CD4+ T lymphocytes after systemicvaccination composed of rDNA prime and live virus vector boostwas minimal and self-limited.

DiscussionIn this study, we established that systemic rDNA priming and virusvector boost can elicit robust immunogen-specific, polyfunctionalCD4+ and CD8+ T lymphocyte responses in breast milk. Interest-

FIGURE 6. Limited SIV-neutralizing and SIVenvelope-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 andNYVAC 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 sys-

temic DNA prime and NYVAC and 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.

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ingly, the polyfunctional cytokine-production profile of immuno-gen-specific breast milk lymphocytes was distinct from that in theblood. This finding likely reflects the regulated trafficking ofvaccine-elicited memory lymphocytes into this mucosal compart-ment or local proliferation of this immunogen-specific lymphocytepopulation within the breast milk compartment. These vaccine-elicited, virus-specific breast milk lymphocytes may be active inthe maternal breast milk compartment and could enhance contain-ment of viruses that are shed in breast milk, such as HIVand CMV.The vaccine-elicited breast milk lymphocytes could also be active inthe infant gastrointestinal tract, and these responses could play a rolein the prevention of mucosally transmitted neonatal pathogens.We previously described a robust virus-specific CD8+ T lym-

phocyte response in breast milk during acute SIV infection thatwas two to three times higher than that in blood (17). Furthermore,studies of HIV/SIV evolution in breast milk suggested that thebreast milk virus population is at least partially populated by virusproduced by locally infected cells, reflected by the frequent oc-currence of groups of genetically identical viruses in breast milkthat are absent or less common in plasma (18–20). Moreover, wereported that breast milk virus quasispecies escape MHC classI-restricted immunodominant CTL responses by mutation of therestricted epitopes at a faster or similar rate to that of the bloodvirus population (17). Therefore, virus replicating in the breastmilk compartment is likely evolving in response to immunepressure from virus-specific CD8+ T lymphocytes. Collectively,these studies suggest that virus-specific CD8+ T lymphocytes acton locally replicating virus in the breast milk compartment. Thus,enhancement of the virus-specific CD8+ T lymphocyte responsesin breast milk through maternal vaccination may contribute toimproved control of virus replication in the breast milk com-partment. Reduction of breast milk virus load may result in a re-duction 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 elicitedby poxvirus vectors (44). High-magnitude CD8+ T lymphocyteresponses and neutralizing Ab responses in milk and blood weregenerated following rAd5 boost, consistent with previous reportsof vaccine-elicited rAd5 responses (24–27). However, the en-hanced CD8+ T lymphocyte and neutralizing Ab responses fol-lowing rAd5 boost may be an additive effect of a second boostadministered over a short time interval. Therefore, elicitation ofdurable, high-magnitude breast milk virus-specific CD8+ T lym-phocyte responses for containment of breast milk virus replicationand neutralizing Ab responses with the ability to block virustransmission in the infant gastrointestinal tract may require morethan 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, 47–49), the predominant breast milk Ab isotype is secretoryIgA. Induction of an SIV Envelope-specific IgA response in breastmilk may play a role in blocking virus transmission in the infantgastrointestinal tract. The vaccine regimen investigated in this studytargets cellular immune responses and, therefore, is not expectedto generate potent mucosal Ab responses. It is possible that anAb-based vaccine regimen, such as a virus vector prime and re-combinant protein boost similar to the regimen that elicited a po-tentially protective Ab response in the recent Thai trial (50), may bemore likely to elicit a mucosal IgA response in breast milk (51).Furthermore, using a mucosal route of immunization may be moreeffective at inducing a mucosal IgA response in breast milk (51–53).

Finally, vaccine-induced activation of CD4+ T lymphocytes hasbeen suggested as a hypothesis for the cause of increased HIVinfections in the vaccine arm of the Step (HIV Vaccine TrialsNetwork) trial evaluating a DNA prime/recombinant adenovirusvector boost HIV vaccine strategy (45, 46). Therefore, we sought toassess the extent of the activation of CD4+ T lymphocytes in milkfollowing systemic vaccination in uninfected monkeys. ActivatedCD4+ T lymphocytes in milk of HIV-infected women may be moreapt at producing virus and interacting with the breastfeeding infant’sgastrointestinal tract. We found minimal and transient increases inthe proportion of CD4+ T lymphocytes in milk expressing mole-cules of activation and the HIV/SIV coreceptor CCR5 followingvaccination in these uninfected, lactating monkeys. A similar pat-tern of limited and transient activation occurred in systemic andcolonic CD4+ T lymphocytes following similar vaccine regimens(41). It is unlikely that these transient and minimal increases in theproportion of activated CD4+ T lymphocytes following vacci-nation would be clinically significant in HIV-infected lactatingwomen, because breast milk lymphocytes are already highly ac-tivated in this setting (54).The demonstration of vaccine-elicited cellular and humoral

immune responses following systemic administration of a candi-date HIV/SIV vaccine regimen provides a platform for furtherdevelopment of maternal vaccine strategies that may impede breastmilk transmission of HIV. Although antiretroviral prophylaxis willbe a mainstay of prevention of HIV transmission via breastfeeding,vaccination could provide a safe and durable adjunctive mechanismof preventing breast milk transmission of HIV. Vaccine strategiesare less reliant on patient compliance and health care infrastructurethan are drug interventions. Furthermore, an effective maternalvaccine administered after delivery would avoid fetal and infanttoxicities. Therefore, it is important to continue to evaluate vaccineregimens well-suited for induction of mucosal responses in breastmilk and assess the efficacy of these interventions in protectionagainst HIV/SIV transmission via breastfeeding in nonhumanprimate models (55) and clinical studies.

AcknowledgmentsWe 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.

DisclosuresThe authors have no financial conflicts of interest.

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