Dan H. Barouch,^1* Sampa Santra,¹ Jörn E. Schmitz,¹ Marcelo J. Kuroda,¹ Tong-Ming Fu,² Wendeline Wagner,³ Miroslawa Bilska,⁴ Abie Craiu,¹ Xin Xiao Zheng,¹ Georgia R. Krivulka,¹ Kristin Beaudry,¹ Michelle A. Lifton,¹ Christine E. Nickerson,¹ Wendy L. Trigona,² Kara Punt,² Dan C. Freed,² Liming Guan,² Sheri Dubey,² Danilo Casimiro,² Adam Simon,² Mary-Ellen Davies,² Michael Chastain,² Terry B. Strom,¹ Rebecca S. Gelman,⁵ David C. Montefiori,⁴ Mark G. Lewis,³ Emilio A. Emini,² John W. Shiver,² Norman L. Letvin¹

With accumulating evidence indicating the importance of cytotoxic T lymphocytes (CTLs) in containing human immunodeficiency virus-1 (HIV-1) replication in infected individuals, strategies are being pursued to elicit virus-specific CTLs with prototype HIV-1 vaccines. Here, we report the protective efficacy of vaccine-elicited immune responses against a pathogenic SHIV-89.6P challenge in rhesus monkeys. Immune responses were elicited by DNA vaccines expressing SIVmac239 Gag and HIV-1 89.6P Env, augmented by the administration of the purified fusion protein IL-2/Ig, consisting of interleukin-2 (IL-2) and the Fc portion of immunoglobulin G (IgG), or a plasmid encoding IL-2/Ig. After SHIV-89.6P infection, sham-vaccinated monkeys developed weak CTL responses, rapid loss of CD4⁺ T cells, no virus-specific CD4⁺ T cell responses, high setpoint viral loads, significant clinical disease progression, and death in half of the animals by day 140 after challenge. In contrast, all monkeys that received the DNA vaccines augmented with IL-2/Ig were infected, but demonstrated potent secondary CTL responses, stable CD4⁺ T cell counts, preserved virus-specific CD4⁺ T cell responses, low to undetectable setpoint viral loads, and no evidence of clinical disease or mortality by day 140 after challenge.

¹ Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215, USA.
² Merck Research Laboratories, West Point, PA 19486, USA.
³ Southern Research Institute, 431 Aviation Way, Frederick, MD 21701, USA.
⁴ Duke University Medical Center, Durham, NC 27710, USA.
⁵ Department of Biostatistical Science, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA.
^*   To whom correspondence should be addressed. E-mail: dan_barouch@hotmail.com

Boosting a DNA-primed immune response with a live recombinant vector has been shown to augment CTL responses and confer control of nonpathogenic viral challenges (13-16). Plasmid IL-2 has also been shown to augment DNA vaccine-elicited immune responses in a variety of murine disease models (17-19). We extended these observations by investigating the vaccine adjuvant properties of IL-2/Ig, a fusion protein consisting of IL-2 and the Fc portion of IgG. This fusion protein has IL-2 functional activity as well as the advantages of divalent avidity and a long in vivo half-life (20, 21).

We previously reported that a plasmid encoding murine IL-2/Ig was able to augment CTL and antibody responses elicited by an HIV-1 gp120 DNA vaccine in mice (22). We have also demonstrated that administration of human IL-2/Ig, either as a protein or a plasmid, markedly augmented DNA vaccine-elicited HIV-1- and SIV-specific immune responses in rhesus monkeys (23). Here, we report that the immunity elicited by IL-2/Ig-augmented DNA vaccines confers control of a highly pathogenic SHIV-89.6P viral challenge.

Vaccine-elicited immune responses. Twenty rhesus monkeys (Macaca mulatta) were immunized with a sham pV1R plasmid (n = 8), SIVmac239 Gag and HIV-1 89.6P Env DNA vaccines alone (n = 4), or these DNA vaccines plus IL-2/Ig (n = 8) (Fig. 1A) (24). The plasmid DNA vaccines expressed genes optimized for high-level expression and used a cytomegalovirus promoter in a pV1R vector backbone. The IL-2/Ig was administered as purified human IL-2/Ig protein in four animals or as a plasmid expressing human IL-2/Ig in four animals. The immune responses elicited by the initial immunizations have been described (23).

In the 15 rhesus monkeys included in this study that expressed the major histocompatibility complex (MHC) class I allele Mamu-A*01, we measured vaccine-elicited CTL responses specific for the Mamu-A*01-restricted immunodominant SIV Gag p11C (CTPYDINQM) and subdominant HIV-1 Env p41A (YAPPISGQI) epitopes (25-28). After the final immunization at week 40, the vaccinated monkeys developed significant circulating p11C- and p41A-specific CD8⁺ T lymphocytes detected by tetramer staining (Fig. 1, B and C) (29, 30). In contrast, the control monkeys had no detectable circulating tetramer-positive CD8⁺ T lymphocytes. Moreover, as expected, since these animals were not vaccinated with an SIV Pol immunogen, none of the monkeys had detectable tetramer-positive CD8⁺ T lymphocytes specific for the Mamu-A*01-restricted SIV Pol p68A epitope (STPPLVRLV) (31). Functional chromium release cytotoxicity assays were also performed with the p11C, p41A, and p68A epitope peptides and corroborated these results (31). The monkeys that received the DNA vaccines plus IL-2/Ig protein or IL-2/Ig plasmid demonstrated markedly augmented vaccine-elicited CTL responses compared with the animals that received the DNA vaccines alone. Because cytokines were only administered with the week 0 and 4 immunizations, the persistence of these augmented CTL responses through week 46 demonstrates the durability of the enhanced CTL priming achieved 10 months earlier. At the time of peak immunity at week 42, the monkeys that received the DNA vaccines plus IL-2/Ig plasmid had an average of 1.2% of circulating CD3⁺CD8⁺ T cells specific for the p11C epitope and 0.5% specific for the p41A epitope. The tetramer-positive CD3⁺CD8⁺ T cells declined thereafter and reached plateau levels of 0.5% for p11C and 0.2% for p41A by the time of challenge. No serum SHIV-89.6P-specific neutralizing antibodies (<1:4 titer) were detected before challenge (31).

Immune responses after viral challenge. Six weeks after the final boost immunization, all 20 rhesus monkeys were challenged by the intravenous route with 100 monkey infectious doses (100 MID₅₀) of cell-free SHIV-89.6P. This virus was derived by in vivo passage of SHIV-89.6, a chimeric virus consisting of the SIVmac239 backbone and the HIV-1 89.6 envelope gene, which was cloned from a primary patient R5/X4 dual-tropic HIV-1 isolate (32-34).

The postchallenge CTL responses specific for the p11C (Fig. 2A), p41A (31), and p68A (31) epitopes were determined by staining freshly isolated peripheral blood mononuclear cells (PBMCs) with tetrameric Mamu-A*01/peptide complexes. The kinetics of the postchallenge CTL responses involved an initial rapid expansion phase that reached a peak at day 14 or 17 after challenge and then fell rapidly to steady-state plateau levels. The control monkeys developed primary p11C-specific CTL responses after challenge, reaching a peak of 1 to 4% of circulating CD3⁺CD8⁺ T cells. In contrast, dramatic secondary CTL responses specific for p11C, reaching a maximum of 18 to 40% of circulating CD3⁺CD8⁺ T cells, were observed in all the animals that received the DNA vaccine plus IL-2/Ig protein or IL-2/Ig plasmid. Intermediate secondary p11C-specific CTL responses were observed in the animals that received the DNA vaccines alone. Augmented secondary CTL responses specific for the p41A epitope were also detected after challenge in the vaccinated monkeys that received IL-2/Ig, but these responses were of a lower magnitude than those specific for p11C (31). Prior to challenge, the p41A-specific CTL responses elicited by DNA vaccination were only 2.5-fold lower in magnitude than the p11C-specific CTL responses. After challenge, however, the p41A-specific responses were 30-fold lower, reflecting the immunodominance of the p11C CTL epitope in Mamu-A*01-positive rhesus monkeys in the setting of SHIV infection. As expected, all the monkeys developed weak primary CTL responses specific for the SIV Pol p68A epitope since none of the monkeys were vaccinated with an SIV Pol immunogen (31).

The virus-specific CD8⁺ T cell responses were further analyzed by interferon- (IFN-) ELISPOT and intracellular IFN- staining assays, the results of which correlated well with the results of the tetramer staining (31). Chromium release cytotoxicity assays and tetramer staining of peptide-stimulated PBMCs (23) further corroborated the results of tetramer staining of freshly isolated PBMCs (Table 1).

Table 1. Analysis of CD8+ T cell responses in the Mamu-A*01-positive monkeys by tetramer staining of freshly isolated PBMCs, tetramer staining of peptide-stimulated PBMCs, and functional chromium release cytotoxicity assays with peptide-stimulated PBMCs on day 63 after challenge (23, 30). Percent CD3+CD8+ cells that bind tetramer are shown for the tetramer assays. Percent-specific lysis at a 5:1 effector-to-target ratio are shown for the cytotoxicity assays. Monkey Tetramer binding (fresh PBMCs) Tetramer binding (stimulated PBMCs) Cytotoxicity assay (stimulated PBMCs) p11C p41A p68A p11C p41A p68A p11C p41A p68A Controls KPB 0.2 0 0.1 1 0 0 3 0 0 KPE 0.8 0 0.1 17 0 1 14 0 0 PKT 0.1 0.1 0 1 0 0 0 0 0 TDE 0.2 0 0 2 1 0 0 0 0 DNA vaccines alone 702 1.5 0.1 0 27 21 1 35 32 2 811 7.9 0 0 14 1 0 14 0 0 820 1.3 0 0.1 18 1 3 16 0 2 DNA + IL-2/Ig protein 712 2.9 0.1 0.1 72 11 4 49 8 3 772 4.4 0.3 0.1 59 28 18 49 26 11 798 3.1 0.3 0.2 63 39 19 43 20 12 839 2.2 0.3 0.1 49 10 4 39 11 2 DNA + IL-2/Ig plasmid 483 5.1 0.4 0.1 88 74 3 55 55 4 728 3.5 0.3 0.1 63 16 8 46 29 2 833 3.0 0.2 0.1 59 22 2 43 19 2 893 4.1 0.5 0.3 86 75 65 54 48 52

Neutralizing antibodies (NAbs) were measured in an MT-2 cell-killing assay, using a stock of SHIV-89.6P expanded in human PBMCs as described (35). Detectable NAb titers were measured in 11 of 12 vaccinated monkeys by day 21 after challenge and in two of eight control monkeys (780 and KPE) by day 28 after challenge (Fig. 2B). All animals that developed detectable NAb responses had comparable peak titers.

Six of the eight control monkeys (all except 780 and KPE) demonstrated a rapid and profound depletion of their CD4⁺ T lymphocytes between days 7 and 21 after challenge, consistent with our previous experience with SHIV-89.6P infection (Fig. 3A) (33, 34). In contrast, all monkeys that received plasmid DNA vaccines plus IL-2/Ig protein or IL-2/Ig plasmid had complete preservation of their CD4⁺ T lymphocytes, with no evidence of declining CD4⁺ T cell counts by day 126 after challenge. At day 70 after challenge, a time by which setpoint of viral replication is reached in SHIV-89.6P-infected rhesus monkeys, a highly significant difference in peripheral blood CD4⁺ T cell counts was evident between the control monkeys and the monkeys that received the DNA vaccines plus IL-2/Ig protein or plasmid [P = 0.00006, by a two-sided Wilcoxon rank sum test (36)]. The monkeys that received the DNA vaccines alone also had detectable CD4⁺ T cell declines, but these were less dramatic than in the control monkeys (P = 0.2). In these animals, a marked but gradual CD4⁺ T cell decline was observed in monkey 811, and small CD4⁺ T cell declines were evident in monkeys 660 and 820. 

To determine whether the challenged monkeys retained functional virus-specific CD4⁺ T lymphocyte activity, we studied their peripheral blood lymphoproliferative responses after stimulation with purified SIV Gag p28 protein (Intracel) and their CD4⁺ T cell IFN- responses after stimulation with SIV Gag and HIV-1 Env peptide pools (37). At day 140 after challenge, all the surviving control monkeys, including the monkeys with preserved total CD4⁺ T lymphocyte counts (780 and KPE), had no detectable lymphoproliferative responses to SIV Gag (stimulation indices < 2) (Fig. 3B). In contrast, all the monkeys that received the IL-2/Ig-augmented DNA vaccines had detectable lymphoproliferative responses to SIV Gag, with stimulation indices of 3.7 to 14.5. One of the monkeys that received the DNA vaccines alone (monkey 702) also had a detectable lymphoproliferative response. The preservation of virus-specific CD4⁺ T lymphocyte function in the vaccinated animals was confirmed by intracellular IFN- staining after stimulation with SIV Gag or HIV-1 Env peptide pools (Table 2).

Table 2. Analysis of CD4+ T cell responses in the surviving monkeys by IFN- intracellular staining assays on day 140 after challenge (37). IFN--positive CD4+ T cells per 106 lymphocytes as measured by intracellular IFN- staining and flow cytometric analysis are shown in response to media control (Mock), SIV Gag peptide pool, HIV-1 Env peptide pool, and staphylococcal enterotoxin B (SEB). Monkeys 766 and KPB had insufficient CD4+ T lymphocytes for analysis. Monkey IFN--positive CD4+ T cells per 106 lymphocytes Mock SIV Gag HIV-1 Env SEB Controls 766 nd nd nd nd 780 167 467 283 20,337 KPB nd nd nd nd KPE 228 411 264 12,470 DNA vaccines alone 660 142 815 519 18,844 702 161 890 325 44,028 811 142 636 346 nd 820 340 1,121 432 11,869 DNA + IL-2/Ig protein 712 164 1,102 410 34,337 772 253 1,353 652 12,352 798 176 1,313 496 3,791 839 141 803 88 14,420 DNA + IL-2/Ig plasmid 483 94 1,250 273 26,726 728 287 692 315 9,993 833 172 1,101 358 16,003 893 218 1,540 400 37,406

Viremia and clinical disease progression. We next measured plasma viral loads in the monkeys by an ultrasensitive branched DNA amplification assay with a detection limit of 400 copies/ml (Bayer Diagnostics). The control monkeys had 1.0 × 10⁷ to 1.8 × 10⁸ copies/ml of virus at the time of peak viremia on day 14 after challenge (Fig. 3C). At day 70 after challenge, six of eight control monkeys had high setpoint viral loads of between 5.0 × 10⁵ and 3.1 × 10⁶ copies/ml. The other two control monkeys had lower setpoint viral loads: 4.6 × 10⁴ copies/ml in monkey KPE and 4.4 × 10² copies/ml in monkey 780. In all the vaccinated monkeys, peak viremia was between 1.2 × 10⁶ and 1.0 × 10⁷ copies/ml, levels significantly lower than in the controls [P = 0.004 and P = 0.008 for monkeys receiving the DNA plus IL-2/Ig or the DNA vaccines alone, respectively, by two-sided Wilcoxon rank-sum tests (36)]. At day 70, setpoint viral loads in the monkeys receiving the IL-2/Ig-augmented DNA vaccines were also significantly lower than in the controls (P = 0.004), and a trend toward reduction in setpoint viral loads was evident in the animals that received the DNA vaccines alone (P = 0.1). At the majority of time points after setpoint, the monkeys that received the DNA vaccines plus IL-2/Ig plasmid had undetectable viremia (<400 copies/ml), although small and transient rises in viremia were seen in monkeys 728 and 893. In the monkeys that received IL-2/Ig protein, viremia was also generally controlled to under 10³ copies/ml. The monkeys that received the DNA vaccines alone had a heterogeneous outcome, with high setpoint viremia in monkey 811, moderate setpoint viremia in monkeys 660 and 820, and controlled viremia in monkey 702.

Compared with the control monkeys, the animals that received the DNA vaccines alone had a 1.9 log reduction in geometric mean viral load after setpoint. The magnitude of this reduction is similar to that seen in previous studies from our laboratory assessing the efficacy of an SIV Gag DNA vaccine (12) or a recombinant MVA-Gag/Pol vaccine (38) in conjunction with SIVsm E660 challenges. The monkeys that received the DNA vaccines plus IL-2/Ig protein or plasmid in the present study had >2.7 and >3.0 log reductions, respectively, in geometric mean viral load after setpoint compared with the control monkeys. In fact, the control of setpoint viremia observed in the monkeys that received the DNA vaccines plus IL-2/Ig plasmid was similar in magnitude to the control of SHIV-89.6P setpoint viremia achieved by immunization with a live, attenuated SIVmac2393 vaccine (39), although the heterologous challenge in that study limits the comparability of results between these reports.

The pathogenicity of the SHIV-89.6P challenge virus was confirmed by the rapid clinical disease progression in the control monkeys that developed low CD4⁺ T lymphocyte counts and high viremia. Seven of the eight control monkeys (all except monkey 780) developed significant clinical disease, and four of these eight monkeys died by day 140 after challenge (Fig. 4 and Table 3). In contrast, all monkeys that received the cytokine-augmented DNA vaccines remained healthy with no documented clinical events or mortality. The prevention of clinical disease in the vaccinated monkeys as compared with the controls was highly significant (P = 0.001, by a two-sided Fisher exact test), and a trend was observed for preventing mortality (P = 0.08). The monkeys that received the DNA vaccines alone had an intermediate clinical outcome, with monkeys 811 and 820 showing signs of clinical disease (P = 0.24 compared with controls).

Table 3. Significant clinical events directly attributable to the SHIV-89.6P infection or the subsequent immunodeficiency in the controls and vaccinated rhesus monkeys through day 140 after challenge. Monkey Disease-attributable clinical events Controls 766 Chronic cough, wound infection, persistent epistaxis 780 None 810 Chronic diarrhea, persistent epistaxis, rash, weight loss, death 826 Fevers, chronic diarrhea, anorexia, death KPB Fevers, persistent epistaxis, chronic diarrhea KPE Chronic diarrhea PKT Fevers, persistent epistaxis, chronic diarrhea, hematochezia, death TDE Explosive diarrhea, weight loss, death DNA vaccines alone 660, 702 None 811 Persistent epistaxis, facial swelling/erythema 820 Persistent epistaxis, facial swelling/erytherma DNA + IL-2/Ig protein 712, 772, 798, 839 None DNA + IL-2/Ig plasmid 483, 728, 833, 893 None

Immune correlates of protection. Scatter plots of data from the Mamu-A*01-positive animals in this study (Fig. 5, A and B) demonstrate significant correlations of prechallenge vaccine-elicited plateau-phase p11C-specific CTL responses determined by tetramer staining, with postchallenge peak p11C-specific CTL responses (P = 0.007, by a two-sided Spearman rank correlation test) and setpoint viral loads (P = 0.04). Levels of peak vaccine-elicited CTLs were less predictive of outcome than those of plateau-phase vaccine-elicited CTLs, the latter presumably reflecting the memory cell population. The asymptotic appearances of the plotted data suggest that a level of vaccine-elicited plateau-phase p11C-specific CTLs may exist (approximately 0.3 to 0.5% of CD3⁺CD8⁺ T cells) above which little additional benefit is discernible after challenge. Similar significant correlations were observed between plateau-phase p41A-specific CTL responses before challenge and peak p41A-specific CTL responses and setpoint viral loads after challenge (31). These results strongly suggest that the improved outcome in animals receiving the cytokine-augmented DNA vaccines resulted from the augmented vaccine-elicited CTL responses in these animals.

No neutralizing antibody (NAb) responses were detected in the vaccinated monkeys prior to challenge, and comparable peak NAb titers developed in the vaccinated monkeys and the two control monkeys with preserved CD4⁺ T lymphocyte counts. The other six control monkeys that rapidly lost their CD4⁺ T lymphocytes would not be expected to generate significant NAb responses (34). Whereas peak CTL responses occurred on day 14 after challenge, coincident with the peak of primary viremia, NAb responses were generally first detectable on day 21 when viremia had already been partially controlled. The initial control of primary viremia in these monkeys therefore appears to be primarily due to CTL activity, as has previously been observed in HIV-infected humans and SIV-infected monkeys (40-42). However, NAbs may also have played a significant role in the control of acute or chronic viremia. In addition, it is possible that IL-2/Ig administration may have modulated nonspecific innate immune functions in these animals and contributed to the containment of virus replication.

Conclusions. In summary, after the SHIV-89.6P challenge, 75% of control animals developed weak immune responses, rapid and profound loss of CD4⁺ T cells, high viral loads, and rapid disease progression. The Mamu-A*01-positive and the Mamu-A*01-negative control monkeys had similar outcomes, and thus this particular MHC class I allele conferred no particular protective or detrimental effect. It is unclear why monkeys 780 and KPE had more favorable outcomes than the other control animals. This may reflect higher virus-specific immune responses or other protective host factors in these animals. The monkeys that received the cytokine-augmented DNA vaccines, in contrast, had uniformly good outcomes with potent immune responses, preserved CD4⁺ T cell counts, low to undetectable viral loads, and no evidence of clinical disease. Moreover, these animals also demonstrated preserved virus-specific CD4⁺ T cell responses, which may confer long-term clinical benefits (43). The monkeys that received the DNA vaccines alone had intermediate and heterogeneous outcomes. Their postchallenge CTL levels reflected both the levels of prechallenge CTLs as well as the levels of viremia driving these responses.

The administration of IL-2/Ig protein or IL-2/Ig plasmid during initial DNA vaccine priming led to augmented immune responses that were capable of controlling viremia and preventing immunodeficiency, clinical disease, and death following a homologous pathogenic SHIV-89.6P challenge in rhesus monkeys. These results raise the possibility that viral replication may also be reduced in humans who have been similarly vaccinated and subsequently infected with HIV-1. As a consequence, such individuals might manifest decreased disease burden and HIV-1 transmission rates (44, 45). Moreover, this strategy of augmenting vaccine-elicited immune responses by cytokine administration should be readily applicable to other vaccine modalities and for other immunotherapeutic purposes.

REFERENCES AND NOTES

1. A. McMichael, Cell 93, 673 (1998) [ISI] [Medline].
2. J. E. Schmitz, et al., Science 283, 857 (1999) [ISI] [Abstract/Full Text].
3. X. Jin, et al., J. Exp. Med. 189, 991 (1999) [ISI] [Abstract/Full Text].
4. L. Musey, et al., N. Engl. J. Med. 337, 1267 (1997) [ISI] [Medline].
5. G. S. Ogg, et al., Science 279, 2103 (1998) [ISI] [Abstract/Full Text].
6. J. A. Wolff, et al., Science 247, 1465 (1990) [ISI] [Medline].
7. D. C. Tang, M. Devit, S. A. Johnson, Nature 356, 152 (1992) [ISI] [Medline].
8. J. B. Ulmer, et al., Science 259, 1745 (1993) [ISI] [Medline].
9. N. L. Letvin, et al., Proc. Natl. Acad. Sci. U.S.A. 94, 9378 (1997) [ISI] [Abstract/Full Text].
10. J. D. Boyer, et al., Nature Med. 3, 526 (1997) [ISI] [Medline].
11. S. Lu, et al., J. Virol. 70, 3978 (1996) [ISI] [Abstract].
12. M. A. Egan, et al., J. Virol. 74, 7485 (2000) [ISI] [Abstract/Full Text].
13. H. L. Robinson, et al., Nature Med. 5, 526 (1999) [ISI] [Medline].
14. S. J. Kent, et al., J. Virol. 72, 10180 (1998) [ISI] [Abstract/Full Text].
15. T. Hanke, et al., J. Virol. 73, 7524 (1999) [ISI] [Abstract/Full Text].
16. T. M. Allen, et al., J. Immunol. 164, 4968 (2000) [ISI] [Abstract/Full Text].
17. M. A. Geissler, A. Gesien, K. Tokushige, J. R. Wands, J. Immunol. 158, 1231 (1997) [ISI] [Abstract].
18. Y.-H. Chow, W.-L. Huang, W.-K. Chi, Y.-D. Chu, M.-H. Tao, J. Virol. 71, 169 (1997) [ISI] [Abstract].
19. K.-Q. Xin, et al., Immunology 94, 438 (1998) [ISI] [Medline].
20. N. F. Landolfi, J. Immunol. 146, 915 (1991) [ISI] [Abstract].
21. P. Nickerson, et al., Transplant. Immunol. 4, 81 (1996) [Medline].
22. D. H. Barouch, et al., J. Immunol. 161, 1875 (1998) [ISI] [Abstract/Full Text].
23. D. H. Barouch, et al., Proc. Natl. Acad. Sci. U.S.A. 97, 4192 (2000) [ISI] [Abstract/Full Text].
24. Large-scale preparations of plasmids were carried out by alkaline lysis and CsCl gradient banding. Rhesus monkeys were vaccinated by intramuscular (i.m.) injections of either 5 mg of HIV-1 89.6P env DNA plus 5 mg of SIVmac239 gag DNA or 10 mg of sham DNA in sterile saline without adjuvant. Half the dose was delivered to each quadriceps muscle in a 0.5-ml volume using a needle-free Biojector apparatus (Bioject). Monkeys were vaccinated at weeks 0, 4, 8, and 40. At weeks 0 and 4, monkeys that received the DNA vaccines alone also received 5 mg of sham DNA 2 days following vaccination (660, 702, 811, 820); monkeys that received human IL-2/Ig protein received 0.5 mg/day affinity-purified protein in two divided doses i.m. on days 1 through 14 following vaccination (712, 772, 798, 839); and monkeys that received human IL-2/Ig plasmid received 5 mg of IL-2/Ig DNA 2 days following vaccination (483, 728, 833, 893). All the vaccinated monkeys except 660 expressed the Mamu-A*01 class I allele. Of the control monkeys, four were Mamu-A*01-negative (766, 780, 810, 826). The four control monkeys that were Mamu-A*01-positive (KPB, KPE, PKT, TDE) were added to the study prior to the week 40 boost immunization. All the animals used in this study were maintained in accordance with the guidelines of the Committee on Animals for Harvard Medical School, the animal care protocols of Southern Research Institute, and the Guide for the Care and Use of Laboratory Animals (National Research Council, National Academy Press, Washington, DC, 1996). After challenge, the immunologic assays, viral load measurements, and clinical care of the animals were performed blinded to the immunizations each monkey received.
25. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; G, Gly; I, Ile; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; and Y, Tyr.
26. M. D. Miller, H. Yamamoto, A. H. Hughes, D. I. Watkins, N. L. Letvin, J. Immunol. 147, 320 (1991) [ISI] [Abstract].
27. T. M. Allen, et al., J. Immunol. 160, 6062 (1998) [ISI] [Medline]. We have simplified previous terminology for the Mamu-A*01-restricted immunodominant SIV Gag epitope from p11C,C-M to p11C for convenience.
28. M. A. Egan, et al., J. Virol. 73, 5466 (1999) [ISI] [Abstract/Full Text].
29. J. D. Altman, et al., Science 274, 94 (1996) [ISI] [Abstract/Full Text].
30. M. J. Kuroda, et al., J. Exp. Med. 187, 1373 (1998) [ISI] [Abstract/Full Text]. Briefly, 1 µg of phycoerythrin-labeled tetrameric Mamu-A*01/peptide complexes was used in conjunction with fluorescein isothiocyanate (FITC)-labeled anti-human CD8 (Leu2a; Becton-Dickinson), ECD-labeled anti-human CD8 (2ST8-5H7; Beckman Coulter), and APC-labeled anti-rhesus monkey CD3 (FN18; Biosource) monoclonal antibodies to stain peptide-specific CD8⁺ T cells. Whole blood (100 µl) from the vaccinated monkeys was directly stained with these reagents, then lysed, washed, and fixed. Samples were analyzed by four-color flow cytometry on a Coulter EPICS Elite ESP system, and gated CD3⁺CD8⁺ T cells were examined for staining with tetrameric Mamu-A*01/p11C, Mamu-A*01/p41A, or Mamu-A*01/p68A complexes.
31. D. H. Barouch et al., data not shown.
32. K. A. Reimann, et al., J. Virol. 70, 3198 (1996) [ISI] [Abstract].
33. K. A. Reimann, et al., J. Virol. 70, 6922 (1996) [ISI] [Abstract].
34. K. A. Reimann, et al., Virology 256, 15 (1999) [ISI] [Medline].
35. J. M. Crawford, et al., J. Virol. 73, 10199 (1999) [ISI] [Abstract/Full Text].
36. Statistical analysis was performed with GraphPad Prism, version 2.01 (GraphPad Software, Inc., 1996). CD4⁺ T lymphocyte counts and viral loads were compared between groups by two-sided Wilcoxon rank-sum tests with Bonferroni adjustments of P values to account for the two major comparisons of each endpoint. Day 70 setpoint values were chosen in order to analyze a complete data set prior to death of any animals. Differences in clinical events and mortality were analyzed by two-sided Fisher exact tests. Correlations of prechallenge vaccine-elicited CTLs and postchallenge peak CTLs or setpoint viral loads were assessed in the vaccinated monkeys by two-sided Spearman rank correlation tests. In all cases, P < 0.05 was considered significant.
37. Intracellular cytokine staining assays were performed as follows. We placed 2 × 10⁶ PBMCs in 17 mm by 100 mm polypropylene tubes containing 1 ml of supplemented RPMI medium, to which 1 µg of each costimulatory antibody (anti-CD28 and anti-CD49d, Becton-Dickinson) was added. Peptide pools were added at a final concentration of 2 µg/ml. Culture tubes were incubated at a 5° slant at 37°C in a humidified 5% CO₂ incubator for 16 hours. Brefeldin A (Sigma) was added for the last 15 hours at a final concentration of 10 µg/ml. Cells were stained with anti-CD3-APC (FN18, Biosource), anti-CD4-PE (OKT4, Ortho Diagnostics), and anti-CD8-PerCP (SK1, Becton-Dickinson) for 30 min and washed with phosphate-buffered saline containing 1% fetal bovine serum. Cells were permeabilized with FACS Permeabilization Buffer (Becton-Dickinson), washed, and stained with anti-IFN--FITC (MD1, Biosource) for 30 min. Samples were fixed in 1% formaldehyde and analyzed on a FACSCalibur flow cytometer (Becton-Dickinson). In order to measure responses of CD4⁺ T lymphocytes, 30,000 gated CD3⁺CD4⁺ lymphocytes were analyzed for intracellular IFN- staining events using CellQuest software (Becton-Dickinson).
38. A. Seth, et al., J. Virol. 74, 2502 (2000) [ISI] [Abstract/Full Text].
39. M. S. Wyand, et al., J. Virol. 73, 8356 (1999) [ISI] [Abstract/Full Text].
40. R. A. Koup, et al., J. Virol. 68, 4650 (1994) [ISI] [Abstract].
41. G. Pantaleo, et al., Nature 370, 463 (1994) [ISI] [Medline].
42. Z. W. Chen, et al., J. Exp. Med. 182, 21 (1995) [ISI] [Abstract].
43. E. S. Rosenberg, et al., Science 278, 1447 (1997) [ISI] [Abstract/Full Text].
44. P. M. Garcia, et al., N. Engl. J. Med. 341, 394 (1999) [ISI] [Medline].
45. T. C. Quinn, et al., N. Engl. J. Med. 342, 921 (2000) [ISI] [Medline].
46. We acknowledge support from NIH grants CA-50139 (N.L.L), AI-85343 (N.L.L. and D.C.M.), AI-65301 (M.G.L.), AI/GF-41521 (T.B.S.), and AI-42298 (T.B.S.). We are grateful to N. Miller, F. Vogel, M. Forman, K. Reimann, W. Lin, A. Miura, R. Kuhnkuhn, C. Lord, J. Frost, T. Steenbeke, C. Crabbs, J. Yalley-Ogunro, N. Persaud, L. Zhu, and J. Joyce for generous advice, assistance, and reagents.

Related articles in Science:

Helminthic Infection and HIV Vaccine Trials.

Miles B. Markus, John E. Fincham;, Dan H. Barouch, and Norman L. Letvin
Science 2001 291: 46-47. (in Letters) [Full Text]  

AIDS:
Enhanced: Preventing AIDS But Not HIV-1 Infection with a DNA Vaccine.

Xuefei Shen and Robert F. Siliciano
Science 2000 290: 463-465. (in Perspectives) [Summary] [Full Text]