A Human T-Cell Leukemia Virus Type 1 Regulatory Element Enhances the Immunogenicity of Human Immunodeficiency Virus Type 1 DNA Vaccines in Mice and Nonhuman Primates

Plasmid DNA vaccines elicit potent and protective immune responsesin numerous small-animal models of infectious diseases. However,their immunogenicity in primates appears less potent. Here weinvestigate a novel approach that optimizes regulatory elementsin the plasmid backbone to improve the immunogenicity of DNAvaccines. Among various regions analyzed, we found that theaddition of a regulatory sequence from the R region of the longterminal repeat from human T-cell leukemia virus type 1 (HTLV-1)to the cytomegalovirus (CMV) enhancer/promoter increased transgeneexpression 5- to 10-fold and improved cellular immune responsesto human immunodeficiency virus type 1 (HIV-1) antigens. Incynomolgus monkeys, DNA vaccines containing the CMV enhancer/promoterwith the HTLV-1 R region (CMV/R) induced markedly higher cellularimmune responses to HIV-1 Env from clades A, B, and C and toHIV-1 Gag-Pol-Nef compared with the parental DNA vaccines. Thesedata demonstrate that optimization of specific regulatory elementscan substantially improve the immunogenicity of DNA vaccinesencoding multiple antigens in small animals and in nonhumanprimates. This strategy could therefore be explored as a potentialmethod to enhance DNA vaccine immunogenicity in humans.

INTRODUCTION

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Introduction

Plasmid DNA vaccines have shown promise as a novel vaccinationmodality based on their simplicity and versatility (31, 32,36). In particular, DNA vaccines can elicit potent and protectivecellular and humoral immune responses in a variety of small-animalmodels (10). However, they have proven substantially less immunogenicin nonhuman primate studies and in clinical trials to date (8,19, 33).

Several approaches have been explored to improve the immunogenicityof DNA vaccines. Our laboratories and others have demonstratedthat the addition of plasmids expressing cytokines and immunomodulatorymolecules can substantially augment DNA vaccine-elicited immuneresponses in both mice and nonhuman primates (3, 4, 15, 16,21, 34, 37). However, the practical requirements of manufacturingand establishing the safety of the plasmid cytokines prior tothe initiation of clinical trials may prove a limitation ofthis strategy (7, 26). Other approaches involve the additionof polymer adjuvants (29) and the use of in vivo electroporationtechniques (24, 35). These strategies have similarly proveneffective in animal models, but their practical utility in clinicaltrials has yet to be demonstrated.

In this study, we investigate a novel strategy involving optimizationof regulatory elements in the backbone of the plasmid DNA vaccine.DNA vaccines often utilize a cytomegalovirus (CMV) enhancer,promoter, and intron to drive high-level expression of a transgenein mammalian cells (32, 38). Here, we explore the effects ofadding the regulatory R region from the 5' long terminal repeat(LTR) of human T-cell leukemia virus type 1 (HTLV-1), whichacts as a transcriptional and posttranscriptional enhancer (30).We find that these CMV/R DNA vaccines elicit substantially higherhuman immunodeficiency virus type 1 (HIV-1)-specific cellularimmune responses compared with the analogous parental DNA vaccinesin both mice and cynomolgus monkeys. Optimization of regulatoryelements thus represents a simple and effective strategy toaugment the immunogenicity of DNA vaccines in primates.

MATERIALS AND METHODS

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Materials and Methods

Plasmid construction. The parental 1012 DNA vaccine plasmid contains the human CMVimmediate early (IE) enhancer, promoter, and intron. To constructthe CMV/R regulatory element, a SacII/HpaI fragment of the 1012plasmid containing the majority of the CMV IE intron was replacedwith a 227-bp EcoRV/HpaI fragment of the HTLV-1 R region (28).The resulting CMV/R plasmid thus contains the human CMV IE enhancer/promoter,followed by the HTLV-1 R region and a 123-bp fragment of CMVIE 3' intron. The splice donor in the R region and the spliceacceptor in the CMV IE 3' intron serve as the pair of splicingsignals. The RSV/R and mUB/R plasmids were similarly constructedby replacing the CMV enhancer/promoter region of the CMV/R plasmidwith a 381-bp AflIII/HindIII fragment of the Rous sarcoma virus(RSV) enhancer/promoter or an 842-bp SpeI/EcoRV fragment ofthe mouse ubiquitin B (mUB) enhancer/promoter, respectively.The mUB enhancer/promoter has been described previously (40).

In vitro expression studies. Murine fibroblast 3T3 cells were transfected with 0.5 µgor parental 1012 (CMV), CMV/R, RSV, RSV/R, mUB, mUB/R DNA vaccinesexpressing HIV-1 Env gp145 ${Delta}$ CFI (9) in six-well plates usingcalcium phosphate; 24 h after transfection, cells were harvestedand lysed in 50 mM HEPES, 150 mM NaCl, 1% NP-40 with proteaseinhibitors and 10 µg total protein was electrophoresedby sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis(PAGE), and gp145 expression was assessed by Western blot analysis.A 1:5,000 dilution of human HIV immunoglobulin G (IgG) was utilizedas the primary antibody, and a 1:5,000 dilution of horseradishperoxidase-conjugated goat anti-human IgG was utilized as thesecondary antibody. The blots were developed with the ECL Westernblot developing system (Amersham Biosciences, Piscataway, NJ).

Animals and immunizations. Six- to 8-week-old BALB/c mice (Charles River Laboratories,Wilmington, MA) were immunized intramuscularly with 50 µgHIV-1 DNA vaccines expressing either clade B Env gp145 ${Delta}$ CFI (9)or a Gag-Pol-Nef fusion protein (13) in 100 µl sterilesaline divided between the right and left quadriceps muscles.Adult cynomolgus monkeys (Bioqual, Rockville, MD) were immunizedintramuscularly with 8 mg of a multivalent HIV-1 DNA vaccinein 1 ml sterile saline delivered by Biojector inoculation intothe right and left quadriceps muscles. This multiclade, multigeneDNA vaccine has been previously described (17) and includesa mixture of plasmids expressing HIV-1 Env gp145 ${Delta}$ CFI from cladesA, B, and C and a clade B Gag-Pol-Nef fusion protein (four plasmidsin a 1:1:1:3 ratio) or these antigens delivered on separateplasmids as individual genes (six plasmids in a 1:1:1:1:1:1ratio). All murine and primate studies were approved by ourInstitutional Animal Care and Use Committees.

ICS assays. CD4⁺ and CD8⁺ T-lymphocyte responses were evaluated by intracellularcytokine staining (ICS) for gamma interferon (IFN- ${gamma}$ ) and tumornecrosis factor alpha (TNF- ${alpha}$ ) as previously described (17). Briefly,splenocytes from immunized mice were harvested and incubatedwith pools of 15-amino-acid peptides overlapping by 11 aminoacids (2.5 µg/ml each) covering the entire HIV-1 Env protein,followed by treatment with 10 µg/ml brefeldin A (Sigma,St. Louis, MO). Cells were then fixed, permeabilized, and stainedusing rat anti-mouse CD3, CD4, CD8, IFN- ${gamma}$ , and TNF- ${alpha}$ monoclonalantibodies (BD Pharmingen, San Diego, CA). The IFN- ${gamma}$ - and TNF- ${alpha}$ positive cells in the CD4⁺ and CD8⁺ cell populations were analyzedwith the program FlowJo (Tree Star, Ashland, OR).

ELISPOT assays. ELISPOT assays were utilized to assess IFN- ${gamma}$ production by murinesplenocytes or monkey peripheral blood mononuclear cells (PBMC)as previously described (4, 5). For cell depletion studies,murine splenocytes were incubated with magnetic microbeads coatedwith anti-CD4 (L3T4) or anti-CD8 (Ly-2) monoclonal antibodies(Miltenyi Biotec, Auburn, CA), and separation using MiniMACScolumns was performed according to the manufacturer's instructions.Cell depletions were approximately 95 to 98% efficient. IFN- ${gamma}$ secretion was then measured in response to overlapping 15-amino-acidpeptides spanning the entire Env, Gag, Pol, or Nef protein.

Statistical analyses. Immunologic data are presented as means with standard errors.Statistical analyses were performed with GraphPad Prism version4.01 (GraphPad Software, Inc., 2004). Comparisons of mean cellularimmune responses between groups of animals were performed bytwo-tailed nonparametric Mann-Whitney tests. In all cases, Pvalues of less than 0.05 were considered significant.

RESULTS

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Results

Optimization of expression from plasmid DNA vaccines in vitro. To develop improved plasmid DNA expression vectors, alternativeregulatory elements were constructed and assessed for theirability to drive in vitro expression of a modified HIV-1 Envprotein, gp145 ${Delta}$ CFI (9). The regulatory R sequence from the 5'LTR of HTLV-1 (12, 30) was incorporated between the CMV enhancer/promoterand splicing acceptor site in the parental 1012 plasmid backboneto construct CMV/R DNA vaccines (Fig. 1A). We also incorporatedthis R element downstream from the Rous sarcoma virus and mousemUB enhancer/promoter elements to create, respectively, RSV/Rand mUB/R DNA vaccines.

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FIG. 1. In vitro expression of HIV-1 Env gp145 ${Delta}$ CFI from various DNA expression vectors. (A). Schematic diagram of the alternative cellular and viral regulatory elements in eukaryotic expression plasmids. (B). Western blot analysis of 3T3 cells transfected with empty parental 1012 (CMV) (lane 1) or those expressing HIV Env gp145 ${Delta}$ CFI with the CMV (lane 2), CMV/R (lane 3), mUB (lane 4), mUB/R (lane 5), RSV (lane 6), or RSV/R (lane 7).

To assess antigen expression from plasmids containing thesetranscriptional regulatory elements, we transfected 3T3 cellswith these expression vectors and measured gp145 ${Delta}$ CFI expressionby Western blots. The expression of gp145 ${Delta}$ CFI from the CMV/Rplasmid was 5- to 10-fold higher than expression from the parental1012 plasmid (Fig. 1B, lanes 2 versus 3). Thus, addition ofthe HTLV-1 R element substantially increased antigen expressiondriven by the CMV promoter. Baseline expression from the mUBplasmid was higher than from the 1012 plasmid but was not furtherenhanced by addition of the R element (Fig. 1B, lanes 4 versus5), suggesting that the effects of adding the R element werepromoter dependent. An increase in expression was observed inthe RSV/R compared to the RSV plasmid (Fig. 1B, lanes 6 versus7). Expression from RSV plasmids is routinely lower than fromthe 1012 plasmid (23).

Immunogenicity of improved expression vectors in mice. We reasoned that enhanced antigen expression may translate toimproved immunogenicity of these novel DNA vaccines in vivo.To explore this possibility, we immunized BALB/c mice (n = 5/group)with 50 µg of the parental 1012 DNA vaccine or the CMV/R,RSV/R, mUB, or mUB/R DNA vaccines expressing HIV-1 Env gp145 ${Delta}$ CFI(9). Mice were immunized three times at weeks 0, 2, and 6. Onday 10 following the final immunization, splenocytes were assessedfor Env-specific cellular immune responses by IFN- ${gamma}$ and TNF- ${alpha}$ intracellular cytokine staining assays. The CMV/R DNA vaccineelicited approximately twofold higher CD4⁺ (P = 0.15) and CD8⁺(P = 0.043) T lymphocyte responses compared with the parental1012 DNA vaccine expressing the same antigen (Fig. 2). In contrast,the RSV/R, mUB, and mUB/R DNA vaccines did not elicit enhancedCD8⁺ immune responses, suggesting that the HTLV-1 R elementselectively improved immunogenicity in the context of the CMVpromoter.

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FIG. 2. Immunogenicity of plasmid DNA vaccines expressing HIV-1 Env gp145 ${Delta}$ CFI in mice. Mice (n = 5/group) were immunized with 50 µg of the parental 1012 DNA vaccine or the CMV/R, RSV/R, mUB, or mUB/R DNA vaccines expressing HIV-1 Env gp145 ${Delta}$ CFI at weeks 0, 2, and 6. After the third immunization, splenocytes were assessed for Env-specific cellular immune responses by IFN- ${gamma}$ and TNF- ${alpha}$ ICS assays. (A) % CD3⁺CD4⁺ IFN- ${gamma}$ /TNF- ${alpha}$ ⁺ and (B) % CD3⁺CD8⁺ IFN- ${gamma}$ /TNF- ${alpha}$ ⁺ splenocytes are shown.

We next compared the immunogenicity of the parental 1012 DNAvaccine and the CMV/R DNA vaccine expressing other antigens.We immunized mice (n = 8/group) with sham plasmids or with theseDNA vaccines expressing the HIV-1 Gag-Pol-Nef fusion protein(13). Mice were immunized twice at weeks 0 and 6, and cellularimmune responses were assessed by IFN- ${gamma}$ ELISPOT assays usingsplenocytes harvested 3 weeks after the initial or boost immunization.Consistent with the prior experiment, we observed approximatelytwofold higher Gag- (P = 0.038) and Pol-specific (P = 0.020)responses elicited by the CMV/R DNA vaccine compared to theparental 1012 DNA vaccine following the initial immunization(Fig. 3A). Nef-specific responses elicited by both plasmidsremained low. Following the boost immunization, responses elicitedby the CMV/R DNA vaccine remained approximately twofold higherthan responses elicited by the parental DNA vaccine using bothunfractionated splenocytes (Fig. 3B) and CD8-depleted splenocytes(Fig. 3C).

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FIG. 3. Immunogenicity of CMV/R plasmid DNA vaccines expressing HIV-1 Gag-Pol-Nef in mice. Mice (n = 8/group) were immunized with 50 µg of the parental 1012 DNA vaccine or the CMV/R DNA vaccine expressing HIV-1 Gag-Pol-Nef at weeks 0 and 6. Splenocytes were assessed for Gag-, Pol-, and Nef-specific cellular immune responses by IFN- ${gamma}$ ELISPOT assays 3 weeks after the (A) initial and (B) week 6 boost immunizations. (C) Splenocytes depleted of CD8⁺ T lymphocytes were also assessed in similar ELISPOT assays following the boost immunization.

Immunogenicity of CMV/R DNA vaccines in cynomolgus monkeys. A number of strategies have improved the immunogenicity of DNAvaccines in inbred strains of mice, but few have been assessedin outbred nonhuman primates (4, 24, 29). We therefore comparedthe immunogenicity of the parental 1012 DNA vaccine with CMV/RDNA vaccine expressing multiple HIV-1 antigens in cynomolgusmonkeys. We immunized two groups of adult cynomolgus monkeys(n = 6/group) with four-plasmid mixtures of 1012 or CMV/R DNAvaccines expressing HIV-1 Env gp145 ${Delta}$ CFI from clades A, B, andC and the Gag-Pol-Nef fusion protein from clade B in a 1:1:1:3ratio. This multiclade, multivalent DNA vaccine has been previouslydescribed (17) and is currently being evaluated in clinicaltrials.

A third group of monkeys was included to investigate whetherseparating the Gag-Pol-Nef fusion protein into separate genesencoded on separate plasmids would further increase immune responsesto these antigens. This third group of monkeys received a six-plasmidmixture of CMV/R DNA vaccines encoding HIV-1 Env gp145 fromclades A, B, and C and separate Gag, Pol, and Nef proteins fromclade B in a 1:1:1:1:1:1 ratio. All monkeys received three immunizationsof 8 mg total DNA vaccine at weeks 0, 4, and 8.

We first compared cellular immune responses against Env cladeA, Env clade B, Env clade C, and Gag, Pol, and Nef from cladeB in monkeys that received the four-plasmid mixtures under thecontrol of CMV (1012) or CMV/R regulatory elements. Monkeysimmunized with the parental 1012 DNA vaccines developed lowand sporadic IFN- ${gamma}$ ELISPOT responses to Env 2 weeks followingthe second immunization at week 6, and no clear responses abovebackground were detected to Gag, Pol, and Nef (Fig. 4A). Incontrast, monkeys immunized with the analogous CMV/R DNA vaccinesexhibited significantly higher responses to all antigens (Fig.4B). Compared to the parental 1012 DNA vaccines, the CMV/R DNAvaccines elicited >10-fold higher ELISPOT responses to Gag(P = 0.0022), Pol (P = 0.0043), and Nef (P = 0.041) and 7- to9-fold higher responses to Env clade A (P = 0.026), B (P = 0.0087),and C (P = 0.030) at this time point. These results demonstratethat the CMV/R DNA vaccines were markedly more immunogenic thanthe parental 1012 DNA vaccines for multiple HIV-1 antigens innonhuman primates.

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FIG. 4. Immunogenicity of multivalent CMV/R DNA vaccines expressing HIV-1 antigens in cynomolgus monkeys. Cynomolgus monkeys (n = 6/group) were immunized with the four-plasmid multivalent (A) 1012 DNA vaccines or (B) CMV/R DNA vaccines expressing HIV-1 Env gp145 ${Delta}$ CFI from clades A, B, and C and a Gag-Pol-Nef fusion protein. (C) A third group of monkeys was immunized with a six-plasmid multivalent CMV/R DNA vaccine expressing HIV-1 Env gp145 ${Delta}$ CFI from clades A, B, and C and separate gag, pol, and nef genes. All monkeys received 8 mg total DNA at weeks 0, 4, and 8. IFN- ${gamma}$ ELISPOT responses were assessed 2 weeks after the second immunization at week 6.

We also observed that separating the Gag-Pol-Nef fusion proteininto individual genes encoded on different plasmids furtherimproved these responses. In particular, monkeys that receivedthe 6-plasmid mixture of CMV/R DNA vaccines developed fourfoldhigher responses to Gag (P = 0.0022), a trend towards twofoldhigher responses to Pol (P = 0.19), and fourfold higher responsesto Nef (P = 0.049) (Fig. 4C), compared to animals that receivedthe four-plasmid mixture of CMV/R DNA vaccines that includedthe Gag-Pol-Nef fusion protein (Fig. 4B). As expected, Env-specificresponses between these two groups of monkeys that receivedthe four-plasmid and six-plasmid mixtures of CMV/R DNA vaccineswere comparable (P = 0.48), since these groups received thesame Env plasmids.

The evolution of mean IFN- ${gamma}$ ELISPOT responses in these groupsof monkeys was evaluated at weeks 0, 2, 6, 10, and 12. Followingthe third DNA immunization at week 8, responses increased inall groups of monkeys (Fig. 5). At week 10, the parental 1012DNA vaccines elicited Env- and Pol-specific responses in themajority of animals, although Gag- and Nef-specific responsesremained low (Fig. 5A). In contrast, the CMV/R DNA vaccineselicited potent and broad responses to all antigens (Fig. 5B and C).At week 10, the four-plasmid CMV/R DNA vaccines (Fig.5B) elicited >10-fold higher ELISPOT responses to Gag (P= 0.0022) and Nef (P = 0.0022), fourfold higher ELISPOT responsesto Pol (P = 0.043), and trends toward 1.5- to 4-fold higherresponses to Env clades A, B, and C (Fig. 5B), compared withthe four-plasmid parental 1012 DNA vaccines (Fig. 5A). Gag-,Pol- and Nef-specific responses remained highest in the animalsthat received the six-plasmid CMV/R DNA vaccines with thesegenes encoded on separate plasmids (Fig. 5C). These studiesconfirm that the CMV/R DNA vaccines elicited substantially higher-magnitudeand broader cellular immune responses to multiple antigens comparedwith the parental 1012 DNA vaccines. Thus, including the HTLV-1R element and separating the gag, pol, and nef genes significantlyenhanced the immunogenicity of HIV-1 DNA vaccines in nonhumanprimates.

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FIG. 5. Mean responses to multivalent CMV/R DNA vaccines expressing HIV-1 antigens in cynomolgus monkeys. (A to C) Cynomolgus monkeys were vaccinated as described in the legend to Fig. 4. Mean and standard errors of IFN- ${gamma}$ ELISPOT responses in these groups of animals were assessed at weeks 0, 2, 6, 10, and 12 following immunization.

DISCUSSION

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Discussion

The relatively low immunogenicity of DNA vaccines in clinicaltrials to date has led to the development of various strategiesto improve the immunogenicity of these vaccines. Approachesthat have proven effective in nonhuman primates include theuse of plasmid cytokines, polymer adjuvants, and in vivo electroporationtechniques (4, 24, 29). A disadvantage of these approaches isthat they require additional materials or devices that add manufacturing,regulatory, and practical complexities that may limit theirlarge-scale clinical application. In this study, we investigatedthe potential utility of a strategy aimed at optimizing regulatoryelements in the plasmid DNA vaccine. We demonstrated that theaddition of the HTLV-1 R element to a CMV expression cassettemarkedly increased DNA vaccine immunogenicity in both mice andcynomolgus monkeys. Importantly, such a modification does notinvolve any additional components or devices and thus does notincrease vaccine complexity. Optimizing immunogenicity by engineeringnovel regulatory elements into the plasmid backbone thereforerepresents a simple, practical, and effective approach thatcould be advanced into clinical trials.

A previous study demonstrated that adding the HTLV-1 R elementdownstream of an SV40 early promoter increased transgene expressionby approximately 10- to 40-fold in vitro (30). The present studyextends this observation by demonstrating that the R elementalso potentiates expression from a strong CMV promoter/enhancer.More importantly, we showed that this enhanced expression translatesinto significantly augmented immune responses in both rodentsand nonhuman primates. We speculate that the beneficial effectsof the R region likely involve recruiting key cellular transcriptionfactors that enhance transgene transcription (12, 39), althoughthe precise mechanism of action remains to be determined. TheR region may also function as an internal ribosome entry site,suggesting that it may act at a posttranscriptional step aswell (2). Alternatively, it is possible that the combinationof CMV enhancer and HTLV-1 R regions stimulate optimal expressionin professional antigen-presenting cells, which would best facilitatethe antigen-specific T-cell response. Interestingly, the RSV/Rand mUB/R DNA vaccines exhibited comparable transgene expressionin vitro compared with CMV/R DNA vaccines (Fig. 1) but neverthelessfailed to result in improved immunogenicity (Fig. 2). Thus,enhanced expression in vitro is not the sole determinant ofenhanced immunogenicity in vivo.

In both mice and cynomolgus monkeys, CMV/R DNA vaccines expressingHIV-1 antigens elicited higher cellular immune responses thanthe parental 1012 DNA vaccines expressing the same antigens.However, the magnitude of the observed effects differed substantiallybetween the two species. While the CMV/R DNA vaccines elicitedonly twofold higher responses in mice (Fig. 3), the CMV/R DNAvaccines elicited >10-fold higher cellular immune responsesto Gag, Pol, and Nef and seven- to ninefold higher responsesto Env after two immunizations in cynomolgus monkeys (Fig. 4and 5). We suspect that this difference may reflect the lowerbaseline immunogenicity of the parental 1012 DNA vaccines innonhuman primates and that the beneficial effects of the R elementmay appear more striking in limiting situations. Consistentwith this observation, the R element had the greatest effectat enhancing the weakest responses elicited by the parental1012 DNA vaccine against Gag and Nef. However, Env- and Pol-specificcellular immune responses were also significantly higher wheninduced by CMV/R DNA vaccines compared with the parental 1012DNA vaccines.

We also observed that the six-plasmid mixture of CMV/R DNA vaccinesthat included Gag, Pol, and Nef on separate plasmids elicitedsignificantly higher cellular immune responses to these antigenscompared to the four-plasmid mixture of CMV/R DNA vaccines thatincluded the Gag-Pol-Nef fusion protein. These effects are particularlynotable since the separate gag, pol, and nef plasmids were eachutilized at one-third the dose of the plasmid encoding the Gag-Pol-Neffusion protein. We speculate that this may reflect enhancedtranslation or mRNA stability of the shorter genes comparedwith the fusion gene, which might potentially affect antigenprocessing and presentation.

A limitation of the current study is that we were not able toassess the protective efficacy afforded by the CMV/R DNA vaccines.Since HIV-1 does not infect cynomolgus monkeys, we could notperform viral challenges in these animals. However, accumulatingdata have confirmed the importance of cellular immune responsesin controlling HIV-1 replication in humans and simian immunodeficiencyvirus replication in rhesus monkeys (14, 22, 27). Moreover,vaccines aimed at eliciting virus-specific cellular immune responseshave afforded partial control of simian-human immunodeficiencyvirus and simian immunodeficiency virus challenges in rhesusmonkeys (1, 4, 11, 18, 20, 25, 29). Thus, we believe that themarkedly increased magnitude and breadth of HIV-1-specific cellularimmune responses afforded by the CMV/R DNA vaccines in nonhumanprimates in the present study will likely prove beneficial inthe development of second-generation DNA vaccines for HIV-1and other pathogens. In particular, incorporating the HTLV-1R element and utilizing separate genes in place of fusion genesrepresent simple and practical strategies to improve DNA vaccines.These strategies could be advanced readily into clinical trials.

ACKNOWLEDGMENTS

We thank Srinivas Rao, Vi Dang, Kristin Beaudry, Faye Yu, KristiMartin, Darci Gorgone, and Michelle Lifton for generous advice,assistance, and reagents.

We acknowledge support from the NIH Vaccine Research Center(G.J.N. and N.L.L.) and NIH grant AI-58727 (D.H.B.).

FOOTNOTES

* Corresponding author. Mailing address: Vaccine Research Center, NIAID, National Institutes of Health, Room 4502, Bldg. 40, MSC-3005, 40 Convent Drive, Bethesda, MD 20892-3005. Phone: 301-496-1852. Fax: 301-480-0274. E-mail: gnabel{at}nih.gov.

These authors contributed equally to this work.

REFERENCES

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