Combination antiretroviral therapy (cART) decreases HIV-related morbidity and mortality1,2 and in some settings, reduces HIV transmission.3–5 Nonetheless, even during successful cART-mediated suppression of HIV viremia, replication-competent viruses persist in certain tissues and long-lived cells,6 and exaggerated systemic inflammation can be readily demonstrated.7–11 This in turn is related to a host of end-organ damage syndromes and to accelerated mortality, particularly in the context of limited CD4+ T-cell reconstitution. Development of HIV-specific cellular immune reactivity, either spontaneously or in response to therapeutic immunization, is accompanied by amelioration of this cascade of events in humans and in nonhuman primate simian immunodeficiency virus infection models.12–24 Thus, induction of such immune responses may confer physiological and clinical benefits in chronic HIV infection, including potential reductions of antiretroviral exposure with its attendant toxicities. Furthermore, identification of an effective strategy to elicit robust HIV-specific immune responses may improve understanding of the biologic correlates of protection against HIV acquisition.
DermaVir is the first in vivo dendritic cell–targeting therapeutic vaccine developed for treatment of HIV-infected persons. The vaccine uses a plasmid DNA (pDNA) immunogen to express 15 HIV antigens, in a synthetic pDNA nanomedicine formulation shown to deliver antigen effectively to lymph node dendritic cells and to induce significant expansions of the HIV-specific precursor/memory T-cell pool.25–32 The effects of DermaVir in humans have been analyzed in 2 previous studies. In a phase 1 study, 9 HIV-infected subjects on suppressive cART who received a single dose (0.1, 0.4, or 0.8 mg of pDNA) of DermaVir demonstrated significant increases in HIV-specific memory T-cell responses after immunization, and these responses were detectable up to a year after vaccine administration.33 In a subsequent phase 1–2 study, 36 cART-naive HIV-infected patients were assigned to 3 different doses of DermaVir or matching placebo. Subjects receiving the intermediate dose of 0.4 mg showed a median plasma HIV decrease from baseline and compared with placebo, of 0.5 log10 copies per milliliter.34 Thus, DermaVir seems to induce immune responses in humans reminiscent of those seen in preclinical nonhuman primate models.35 A5176 was designed to extend these observations to a larger group of HIV-infected subjects receiving suppressive cART.
Study Design and Study Population
A5176 was a multicenter, randomized, placebo-controlled clinical trial comparing 3 doses of DermaVir and a placebo arm among chronically HIV-infected subjects of both sexes, aged 18–50 years, receiving stable cART, with confirmed plasma HIV RNA of <50 copies per milliliter, CD4+ T-cell count of >350 cells per microliter, nadir CD4+ T-cell count of >250 cells per microliter, and negative hepatitis B surface antigen and hepatitis C antibody. Other exclusion criteria are listed in Table S1 (see Supplemental Digital Content, http://links.lww.com/QAI/A460). Participants were recruited from University of California Davis Medical Center (Sacramento, CA), Chicago Children's Hospital (Chicago, IL), Metro Health Medical Center (Cleveland, OH), University Hospitals of Cleveland Case Medical Center (Cleveland, OH), and University of Pittsburgh (Pittsburgh, PA). The protocol was approved by the participating institutions' institutional review boards. All subjects provided written informed consent.
Subjects were sequentially enrolled into 1 of 3 dosing cohorts and randomized to receive DermaVir (6 subjects per cohort) or placebo (2 subjects per cohort). DermaVir or placebo was administered over either 2 or 4 skin sites on the left and right upper back and both upper ventral thighs using the DermaPrep device.33 The selected area was disinfected and exfoliated to disrupt the stratum corneum of the skin and induce mild erythema. This procedure enhances DermaVir nanoparticle endocytosis by activated Langerhans cells and delivery of pDNA-encoded antigens to naive T-lymphocytes in the regional lymph nodes.26 After skin preparation, a patch covering approximately 80 cm2 was applied over the area, and the vaccine solution was placed on the skin underneath the patch with a needleless syringe. The patch was kept for 3 hours after vaccination. Subjects assigned to the active arm received the following doses of DermaVir: In the low-dose cohort, 3 vaccinations (0.1 mg of DNA per subject, 0.8 mL of total over 2 skin sites of 80 cm2 each, 0.4 mL per site) on weeks 1, 7, and 13; in the intermediate-dose cohort, 3 vaccinations (0.4 mg of DNA per subject, 3.2 mL of total over 4 skin sites of 80 cm2 each, 0.8 mL per site) on study weeks 1, 7, and 13; in the high-dose cohort, 6 vaccinations (0.4 mg of DNA per subject, 3.2 mL of total over 4 skin sites of 80 cm2 each, 0.8 mL per site) on study weeks 0, 1, 6, 7, 12, and 13. Subjects in the placebo arm received a matching glucose placebo, administered as described above. Each cohort was enrolled sequentially from low to high dose. Enrollment into each higher dose cohort required that at least 6 of the 8 subjects in the previous dose cohort had been followed on study for ≥14 days after their second study vaccination and that no subject had experienced a primary safety endpoint as defined below.
Assessment of Safety Study Endpoints
The primary endpoint was the occurrence of grade 3 or higher clinical or laboratory adverse events possibly or definitely related to study treatment from first day of study treatment to 28 days after last study vaccination. Events were defined and graded according to the current Division of AIDS Adverse Event Grading Table (available at: http://rsc.tech-res.com/safetyandpharmacovigilance). Relationship to study treatment was determined by the protocol core team, including site clinicians, blinded to treatment arm. Reactions felt to be solely due to adhesive and not the vaccine itself were not used to determine the primary safety endpoint. Antidouble–stranded DNA was measured by enzyme-linked immunosorbent assay to exclude potential autoimmune reactions to the immunization.
Assessment of Immunogenicity Endpoints
We report the results of a previously described36 extended culture interferon (IFN)-γ enzyme-linked immunosorbent spot (ELISPOT) assay as the main immunogenicity readout. The ELISPOT readout was chosen over other immunogenicity endpoints because of its reproducibility and sensitivity across HIV vaccine studies.37 This assay detects predominantly T-cell precursors with high proliferating capacity (PHPC), a memory T-cell population that has been shown to be induced by DermaVir immunization and to correlate inversely with plasma viremia in untreated HIV infection.36
Briefly, cryopreserved peripheral blood mononuclear cells (PBMCs) were thawed, rested overnight, and suspended in R10 complete culture medium to a final concentration of 5 × 105 cells per milliliter. Cases in which cell viability at the beginning of the culture was below 80% were excluded from the analysis. The cutoff of 80% was chosen to remove the artifactual effect of poor viability at the start of the PHPC culture (24 hours after thawing) on the PHPC readout. Cells were plated in 48-well tissue culture plates with the HIV peptide pools, phytohemaglutinin/staphylococcal enterotoxin B as positive control or medium alone as negative control and were cultured at 37°C in 5% of CO2 for 12 days. On days 3 and 7, 500 μL of supernatant per well was removed and replaced with fresh complete culture medium supplemented with 10 IU/mL recombinant human interleukin (IL)-2. The HIV peptides were obtained from the NIH AIDS Research and Reference Reagent Program and were 15 amino acids in length overlapping by 11, representing the entire HIV-1 subtype B sequence of Gag (divided into 3 pools, p17, p24, and p15), Tat, and Rev. At the end of the 12-day culture, cells were harvested, and a standard overnight ELISPOT assay was performed as previously described with minor modifications.38 Briefly, the cells were plated in triplicate into 96-well antihuman INF-γ antibody precoated ELISPOT plates (BD Biosciences, San Diego, CA) at 2.5 × 104 cells per well and stimulated with each of the HIV peptide pools (5 μg/mL), phytohemaglutinin/staphylococcal enterotoxin B (5 μg/mL), or medium alone. After incubation for 24 hours at 37°C in 5% of CO2, plates were washed and developed following the manufacturer's protocol (BD Biosciences). Spots were counted using an automated ELISPOT reader. Results were expressed as net IFNγ spot-forming units (SFU) per million PBMCs (the difference between mean SFU/million PBMCs in stimulated wells and mean SFU/million PBMCs in medium-only wells) multiplied by the proliferation index (number of stimulated cells/number of control-stimulated cells at the end of the culture) to account for the expected expansion of T cells during prolonged culture.
Because HIV-specific T-cell responses were frequently detected at baseline, we defined the development of a new response in 2 ways: (1) as any postvaccination response that was >1 log10 above the log10 baseline response, regardless of study arm and (2) as an area under the response curve (AUC) to a given peptide antigen pool that was greater than the maximum AUC to the same stimulant observed in the placebo arm, which meant that this definition applied only to those subjects assigned to a DermaVir dosing group. All time points were considered, and the point of greatest response magnitude was derived from the data.
Other immunogenicity assays included HIV-specific CD4+ and CD8+ T-cell proliferation by carboxyfluorescein succinimidyl ester (CFSE) dye dilution, HIV-specific intracellular cytokine (IFN-γ and IL-2) staining by flow cytometry, and conventional overnight ELISPOT. As previously reported,36,39 these assays were less sensitive than the cultured ELISPOT assay and will not be reported here.
Vaccine site reactions, clinical events, signs and symptoms, laboratory toxicities, and adverse events grade 2 or higher from first day of study treatment to 28 days after the last study vaccination were tabulated by study group considering all the subjects who started study treatment. Time-averaged AUC was computed using the linear trapezoidal method only for subjects who started study treatment with nonmissing cultured ELISPOT data until week 37 (ie, AUC/37). Differences among study groups were analyzed by Fisher exact test or Wilcoxon rank-sum test as appropriate. Dose–response trends were assessed by the Jonckheere–Terpstra test. Changes from baseline were assessed by the sign test. All P values presented were 2-sided and nominal unadjusted for multiple comparisons. Analyses were performed using SAS, version 9.2 (SAS Institute, Cary, NC) and StatXact 8 PROCs (Cytel, Cambridge, MA).
Twenty-six subjects were enrolled. One subject in the intermediate-dose group withdrew voluntarily before receiving any study vaccination and was excluded from all analyses. Another subject was noncompliant with study visits and was discontinued at week 8. Both the subjects were replaced. Groups were well balanced with respect to baseline characteristics, except for a slightly younger age in the placebo group. Detailed demographic characteristics are shown in Table 1. Median age was 39 years, median baseline CD4 count was 645 cells per microliter, 92% of participants were men, and 64% were white. All had plasma HIV RNA <50 copies per milliliter at study entry.
Overall, study interventions were well tolerated. No subject experienced a grade ≥3 treatment-related adverse event (primary endpoint). There were no deaths. The overall proportions of subjects experiencing any adverse event between the first treatment date and 28 days after the last dose were 57% in the placebo arm and 67%, 100%, and 50% in each of the low-, intermediate-, and high-dose DermaVir groups, respectively (P = 0.31). Differences were also not significant comparing the placebo with the combined DermaVir arms (P = 0.64). General body complaints (aches/pain/discomfort, asthenia/fatigue/malaise, and fever) were most common and occurred in 7 subjects, followed by cutaneous symptoms (pruritus and rash) and blood chemistry abnormalities in 5 subjects each. A summary of adverse events is shown in Table 2. The proportion of treatment-related adverse events was comparable between the DermaVir and placebo arms (0/7, 3/6, 1/6, and 0/6 in the placebo arm and the low-, intermediate-, and high-dose DermaVir groups, respectively, P = 0.063). All adverse events felt to be possibly, probably, or definitely related to study agent and all abnormal blood chemistries were mild (grade 1–2) and no subject had to modify or discontinue the study treatment due to an adverse event. None of the subjects with available data developed detectable antidouble–stranded DNA antibodies.
Changes in CD4+ and CD8+ T Cells
CD4+ and CD8+ T-cell counts were measured at weeks 0, 3, 9, 15, 17, 24, 37, and 61 and did not change significantly during the study in any of the dosing groups (P ≥ 0.688 and ≥0.219, respectively). Median [interquartile range (IQR)] change in CD4+ T-cell count from baseline to week 61 was +13 (−132 to +229) cells per microliter in the combined DermaVir groups versus +151 (−126 to +237) in the placebo group. Differences between the DermaVir arm (either combined or each group separately) and the placebo arm were not statistically significant at any of the measured time points, and there was no evidence for a dose-related trend in CD4+ T-cell change across the dosing groups of DermaVir (Jonckheere–Terpstra test P values for all time points >0.49).
Changes in Plasma HIV RNA
All subjects had plasma HIV RNA below the limit of detection at baseline and all maintained virological suppression through week 24, except for 3 subjects who had episodes of transient viremia (1 subject in each of the DermaVir groups, with 65 copies at week 9, 274 copies at week 24, and 933 copies at week 61, respectively).
Extended Culture ELISPOT (PHPC) Responses
Cultured ELISPOT responses are shown in Figure 1. There was considerable baseline variability in all groups. Overall, there was a trend toward higher responses in the DermaVir groups, particularly the low- and intermediate-dose groups, compared with placebo. Responses in the high-dose group were not significantly different than the placebo group at any of the studied time points in response to any of the peptide pools used (all P > 0.05), and responses in this group did not correlate significantly with baseline CD4+ T-cell count. At week 17 (4 weeks after last study vaccination), responses to Gag p24 were significantly greater among subjects in the intermediate-dose group compared with control subjects [median (IQR): 67,600 (5633–74,368) vs. 1,194 (9–1667) net SFU per million cells, P = 0.032]. Responses to both Gag p24 and Gag p15 among intermediate-dose subjects were also significantly greater than among high-dose subjects at week 17 [67,600 (5633–74,368) vs. 485 (172–1440), P = 0.017 and 17,067 (5495–67,600) vs. 642 (74–4086) net SFU per million cells, P = 0.03].
The increased anti-HIV T-cell response frequency in the intermediate-dose group was consistent across stimulus conditions (Fig. 1): with the exception of responses to Gag p15 at week 37, responses in the intermediate-dose group were higher than those in the placebo group and the other 2 DermaVir dosing groups at all postvaccine time points (Fig. 1), although the difference was only significant at week 17.
Because baseline reactivity showed substantial variation, we tested whether there was evidence for a change from baseline in cultured ELISPOT responses in any of the groups (not shown). Response to Gag p15 increased significantly among participants in the high-dose group from baseline to week 17 [median (IQR) change 149 (44–3049) net SFU/million, P = 0.031], but this increase was not significantly different from placebo (P = 0.247). Participants in the intermediate-dose group experienced a marginally significant increase from baseline at the same time point in response to Gag p15 [2859 (1867–56,933), P = 0.06], and this change was significantly greater than in the placebo group [0 (−713 to 297), P = 0.016].
We observed a similar trend toward a greater absolute week 0–37 AUC in the intermediate-dose group compared with placebo in response to Gag p24, Gag p15, and Tat-Rev (Fig. 1), although these differences were marginal (all P = 0.057). In this analysis, the intermediate-dose group also showed significantly greater responses to each of those peptide pools compared with the high-dose group (P = 0.019, 0.019, and 0.038, respectively).
Finally, we compared the proportions of subjects who achieved a response to the study intervention in each of the groups. When a response was defined as a 1 − log10 net SFU increase from baseline to each of the Gag peptide pools, 20%–60% of the DermaVir recipients were classified as responders compared with 20% of the placebo recipients, although the differences were not statistically significant (Table 3). A similar trend was observed when we defined a DermaVir-induced response as an AUC in any DermaVir dosing group that was higher than the AUC in the placebo group. In the latter analysis, 100% of subjects in the low- and intermediate-dose groups achieved a response compared with only 33% of subjects in the high-dose group (Table 3).
An effective therapeutic vaccine capable of inducing durable HIV-specific CD8+ T-cell responses with potent cytotoxic activity and reducing viremia could theoretically confer many of the benefits of cART without some of its limitations. Evidence in favor of this approach comes from observations in nonhuman primate models indicating that cytotoxic T-cell responses are crucial in maintaining spontaneous and DermaVir-induced control of viremia15,35 by the recognized persistence of vigorous CD8+ T-cell responses in human long-term nonprogressors40–42 and by the overrepresentation of protective human leukocyte antigen (HLA) alleles thought to be capable of presenting highly conserved HIV epitopes among spontaneous controllers of HIV disease progression.43 Previous human studies have shown trends toward enhanced immune responses and virological control after administration of various cytotoxic T-lymphocyte (CTL)-targeting therapeutic vaccination strategies.33,44–52 Furthermore, in vitro studies indicate that Gag peptide stimulation of CD8+ T-cells results in efficient killing of autologous HIV-infected CD4+ targets reactivated from latency, suggesting that induction of CTL responses may be a component in a successful reservoir eradication strategy.53
In A5176, we administered escalating doses of DermaVir, a therapeutic vaccine candidate, or matched placebo to 25 HIV-infected patients with chronic HIV infection receiving suppressive cART. The study interventions, including repeated doses of DermaVir, were well tolerated, without dose-limiting toxicities. Adverse events were generally mild, and there was no significant difference in adverse event frequency among study groups (Table 2).
DermaVir administration tended to be associated with greater cultured ELISPOT (PHPC) responses to all 3 Gag peptide pools compared with placebo, particularly among patients who received the intermediate dose of DermaVir. These DermaVir-induced T-cell responses were greatest at week 17, 4 weeks after the last immunization dose. Responses to Tat/Rev, on the other hand, were infrequently observed and of low magnitude (Fig. 1), perhaps due to the smaller number of potential high-affinity epitopes in these small proteins.30 Although the large number of comparisons included in the analysis could conceivably have led to spurious results, these findings are consistent with a previous study in treatment-naive patients, in which the 0.4 mg of dose was associated with a 0.5 log reduction in plasma HIV RNA and with the most robust cultured ELISPOT responses at 24 weeks.34 The greater immunogenicity of the intermediate dose may reflect high-dose antigen-induced hyporesponsiveness as shown in animal models.54
The nature of the T-cell responses elicited in this study deserves separate comment. Although T-cell subsets were not sorted or characterized in this study, the cultured ELISPOT assay detects predominantly antigen-specific long-lived central memory T cells,55,56 and robust central memory T-cell responses have been associated with spontaneous control of HIV viremia.57 Thus, DermaVir could enhance the type of cellular immune responses needed for control of plasma viremia, which are almost universally lost early in the course of HIV infection.
Nonetheless, the magnitude of DermaVir-induced responses in this study was variable and reached statistical significance relative to placebo only at week 17 for the intermediate-dose group and only in response to certain peptide pools. A number of limitations in the study design may have contributed to these findings. First, the small sample sizes in each of the dosing groups hindered detection of significant differences, despite responses in the DermaVir groups that were thousands of net SFUs above those in the placebo group (Fig. 1). Second, in this as in most HIV vaccine studies, predicting and monitoring the breadth and magnitude of meaningful HIV-specific responses remain a major challenge because the range of immunodominant epitopes varies dramatically from person to person because of the complex sequence of mechanisms that enable the establishment of immunodominance.58 Indeed, an analysis of the high-affinity epitopes present in DermaVir predicted that this immunogen can potentially present up to 933 high-affinity major histocompatibility complex class (MHC) class I-restricted epitopes and up to 2330 high-affinity MHC class II-restricted epitopes.30 By comparison, the peptide pools used in our monitoring assay cover only approximately 25% of the entire repertoire potentially presented by DermaVir. Therefore, a more closely matched set of peptides may be needed to test the full range of immunogenicity induced by this candidate preparation. Equally important will be to assess the HLA background of future study participants and to map in greater detail the epitopes predicted to be bound with highest affinity.
This short-term study was not designed to assess efficacy, as neither clinical nor virological endpoints were included in the study design, but possible roles for this therapeutic vaccine strategy can nonetheless be envisaged. If, as shown previously,34 DermaVir is capable of reducing plasma HIV RNA durably and safely in the absence of cART, this approach could allow delayed initiation or even interruption of antiretroviral therapy in some individuals, or it could be used as a bridge strategy for patients awaiting new antiretroviral options in the setting of multidrug resistance or intractable toxicity. Therapeutic immunization could also be a component of a future multipronged approach to eradicate HIV infection.
In summary, DermaVir is a safe candidate therapeutic vaccine that may induce HIV-specific central memory T-cell responses in some HIV-infected persons receiving suppressive cART. Larger studies, potentially including DermaVir immune intensification on cART before activation of latently infected cells followed by an analytic treatment interruption, are necessary to quantify the entire repertoire of potential responses more accurately, to explore the clinical, virological, and immunological effects of repeated administration, and to define the potential therapeutic role of DermaVir in the HIV armamentarium.
The authors thank X.-L. Huang, W. Jiang, and P. Zhang from the University of Pittsburgh for their technical assistance with the cultured ELISPOT assay.
1. Palella FJ Jr, Delaney KM, Moorman AC, et al.. Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. HIV Outpatient Study Investigators. N Engl J Med. 1998;338:853–860.
2. UNAIDS. World AIDS Report 2011. Geneva, Switzerland; 2011.
3. Das M, Chu PL, Santos GM, et al.. Decreases in community viral load are accompanied by reductions in new HIV infections in San Francisco. PLoS One. 2010;5:e11068.
4. Wood E, Kerr T, Marshall BD, et al.. Longitudinal community plasma HIV-1 RNA concentrations and incidence of HIV-1 among injecting drug users: prospective cohort study. BMJ. 2009;338:b1649.
5. Cohen MS, Chen YQ, McCauley M, et al.. Prevention of HIV-1 infection with early antiretroviral therapy. N Engl J Med. 2011;365:493–505.
6. Wong JK, Hezareh M, Gunthard HF, et al.. Recovery of replication-competent HIV despite prolonged suppression of plasma viremia. Science. 1997;278:1291–1295.
7. El-Sadr WM, Lundgren JD, Neaton JD, et al.. CD4+ count-guided interruption of antiretroviral treatment. N Engl J Med. 2006;355:2283–2296.
8. Kuller LH, Tracy R, Belloso W, et al.. Inflammatory and coagulation biomarkers and mortality in patients with HIV infection. PLoS Med. 2008;5:e203.
9. Lederman MM, Calabrese L, Funderburg NT, et al.. Immunologic failure despite suppressive antiretroviral therapy is related to activation and turnover of memory CD4 cells. J Infect Dis. 2011;204:1217–1226.
10. Neuhaus J, Jacobs DR Jr, Baker JV, et al.. Markers of inflammation, coagulation, and renal function are elevated in adults with HIV infection. J Infect Dis. 2010;201:1788–1795.
11. Marin B, Thiebaut R, Bucher HC, et al.. Non-AIDS-defining deaths and immunodeficiency in the era of combination antiretroviral therapy. AIDS. 2009;23:1743–1753.
12. Abdel-Motal UM, Gillis J, Manson K, et al.. Kinetics of expansion of SIV Gag-specific CD8+ T lymphocytes following challenge of vaccinated macaques. Virology. 2005;333:226–238.
13. Baker BM, Block BL, Rothchild AC, et al.. Elite control of HIV infection: implications for vaccine design. Expert Opin Biol Ther. 2009;9:55–69.
14. Betts MR, Nason MC, West SM, et al.. HIV nonprogressors preferentially maintain highly functional HIV-specific CD8+ T cells. Blood. 2006;107:4781–4789.
15. Friedrich TC, Valentine LE, Yant LJ, et al.. Subdominant CD8+ T-cell responses are involved in durable control of AIDS virus replication. J Virol. 2007;81:3465–3476.
16. Hel Z, Nacsa J, Tryniszewska E, et al.. Containment of simian immunodeficiency virus infection in vaccinated macaques: correlation with the magnitude of virus-specific pre- and postchallenge CD4+ and CD8+ T cell responses. J Immunol. 2002;169:4778–4787.
17. Mudd PA, Watkins DI. Understanding animal models of elite control: windows on effective immune responses against immunodeficiency viruses. Curr Opin HIV AIDS. 2011;6:197–201.
18. Nilsson C, Makitalo B, Thorstensson R, et al.. Live attenuated simian immunodeficiency virus (SIV)mac in macaques can induce protection against mucosal infection with SIVsm. AIDS. 1998;12:2261–2270.
19. Pereyra F, Jia X, McLaren PJ, et al.. The major genetic determinants of HIV-1 control affect HLA class I peptide presentation. Science. 2010;330:1551–1557.
20. Reynolds MR, Weiler AM, Weisgrau KL, et al.. Macaques vaccinated with live-attenuated SIV control replication of heterologous virus. J Exp Med. 2008;205:2537–2550.
21. Saez-Cirion A, Lacabaratz C, Lambotte O, et al.. HIV controllers exhibit potent CD8 T cell capacity to suppress HIV infection ex vivo and peculiar cytotoxic T lymphocyte activation phenotype. Proc Natl Acad Sci U S A. 2007;104:6776–6781.
22. Schmitz JE, Kuroda MJ, Santra S, et al.. Control of viremia in simian immunodeficiency virus infection by CD8+ lymphocytes. Science. 1999;283:857–860.
23. Turnbull EL, Lopes AR, Jones NA, et al.. HIV-1 epitope-specific CD8+ T cell responses strongly associated with delayed disease progression cross-recognize epitope variants efficiently. J Immunol. 2006;176:6130–6146.
24. Wyand MS, Manson K, Montefiori DC, et al.. Protection by live, attenuated simian immunodeficiency virus against heterologous challenge. J Virol. 1999;73:8356–8363.
25. Lisziewicz J, Kelly L, Lori F. Topical DermaVir vaccine targeting dendritic cells. Curr Drug Deliv. 2006;3:83–88.
26. Lisziewicz J, Trocio J, Whitman L, et al.. DermaVir: a novel topical vaccine for HIV/AIDS. J Invest Dermatol. 2005;124:160–169.
27. Lori F. DermaVir: a plasmid DNA-based nanomedicine therapeutic vaccine for the treatment of HIV/AIDS. Expert Rev Vaccines. 2011;10:1371–1384.
28. Lori F, Trocio J, Bakare N, et al.. DermaVir, a novel HIV immunisation technology. Vaccine. 2005;23:2030–2034.
29. Lorincz O, Toke ER, Somogyi E, et al.. Structure and biological activity of pathogen-like synthetic nanomedicines. Nanomedicine. 2012;8:497–506.
30. Somogyi E, Xu J, Gudics A, et al.. A plasmid DNA immunogen expressing fifteen protein antigens and complex virus-like particles (VLP+) mimicking naturally occurring HIV. Vaccine. 2011;29:744–753.
31. Lisziewicz J, Gabrilovich DI, Varga G, et al.. Induction of potent human immunodeficiency virus type 1-specific T-cell-restricted immunity by genetically modified dendritic cells. J Virol. 2001;75:7621–7628.
32. Toke ER, Lorincz O, Somogyi E, et al.. Rational development of a stable liquid formulation for nanomedicine products. Int J Pharm. 2010;392:261–267.
33. Lisziewicz J, Bakare N, Calarota SA, et al.. Single DermaVir immunization: dose-dependent expansion of precursor/memory T cells against all HIV antigens in HIV-1 infected individuals. PLoS One. 2012;7:e35416. doi: 10.1371/journal.pone.0035416.
34. van Lunzen J, Pollard R, Stellbrink HJ, et al.. DermaVir for initial treatment of HIV-infected subjects demonstrates preliminary safety, immunogenicity and HIV-RNA reduction versus placebo immunization. Paper presented at: XVIII International AIDS Conference, Abstract CDB0237; July 18–23, 2010; Vienna, Austria.
35. Lisziewicz J, Trocio J, Xu J, et al.. Control of viral rebound through therapeutic immunization with DermaVir. AIDS. 2005;19:35–43.
36. Calarota SA, Foli A, Maserati R, et al.. HIV-1-specific T cell precursors with high proliferative capacity correlate with low viremia and high CD4 counts in untreated individuals. J Immunol. 2008;180:5907–5915.
37. Gill DK, Huang Y, Levine GL, et al.. Equivalence of ELISpot assays demonstrated between major HIV network laboratories. PLoS One. 2010;5:e14330 doi: 10.1371/journal.pone.0014330.
38. Huang XL, Fan Z, Kalinyak C, et al.. CD8(+) T-cell gamma interferon production specific for human immunodeficiency virus type 1 (HIV-1) in HIV-1-infected subjects. Clin Diagn Lab Immunol. 2000;7:279–287.
39. Goonetilleke N, Moore S, Dally L, et al.. Induction of multifunctional human immunodeficiency virus type 1 (HIV-1)-specific T cells capable of proliferation in healthy subjects by using a prime-boost regimen of DNA- and modified vaccinia virus Ankara-vectored vaccines expressing HIV-1 Gag coupled to CD8+ T-cell epitopes. J Virol. 2006;80:4717–4728.
40. Klein MR, van Baalen CA, Holwerda AM, et al.. Kinetics of Gag-specific cytotoxic T lymphocyte responses during the clinical course of HIV-1 infection: a longitudinal analysis of rapid progressors and long-term asymptomatics. J Exp Med. 1995;181:1365–1372.
41. Pantaleo G, Demarest JF, Schacker T, et al.. The qualitative nature of the primary immune response to HIV infection is a prognosticator of disease progression independent of the initial level of plasma viremia. Proc Natl Acad Sci U S A. 1997;94:254–258.
42. Rinaldo C, Huang XL, Fan ZF, et al.. High levels of anti-human immunodeficiency virus type 1 (HIV-1) memory cytotoxic T-lymphocyte activity and low viral load are associated with lack of disease in HIV-1-infected long-term nonprogressors. J Virol. 1995;69:5838–5842.
43. Dinges WL, Richardt J, Friedrich D, et al.. Virus-specific CD8+ T-cell responses better define HIV disease progression than HLA genotype. J Virol. 2010;84:4461–4468.
44. Schooley RT, Spritzler J, Wang H, et al.. AIDS clinical trials group 5197: a placebo-controlled trial of immunization of HIV-1-infected persons with a replication-deficient adenovirus type 5 vaccine expressing the HIV-1 core protein. J Infect Dis. 2010;202:705–716.
45. Routy JP, Boulassel MR, Yassine-Diab B, et al.. Immunologic activity and safety of autologous HIV RNA-electroporated dendritic cells in HIV-1 infected patients receiving antiretroviral therapy. Clin Immunol. 2010;134:140–147.
46. Connolly NC, Whiteside TL, Wilson C, et al.. Therapeutic immunization with human immunodeficiency virus type 1 (HIV-1) peptide-loaded dendritic cells is safe and induces immunogenicity in HIV-1-infected individuals. Clin Vaccine Immunol. 2008;15:284–292.
47. Rockstroh JK, Pantaleo G, Van Lunzen J, et al.. A Phase IIB, Randomized, double-blind, multicenter, immunogenicity study of Vacc-4x versus placebo in HIV-1-infected patients. Paper presented at: AIDS Vaccine 2011, Abstract P1821 LB; September 12, 2011; Bangkok, Thailand.
48. Vardas E, Stanescu I, Leinonen M, et al.. Indicators of therapeutic vaccine effect using multi-HIV B clade DNA in treatment-naïve subtype C HIV-1 infected subjects. Paper presented at: AIDS 2010, Abstract MOPD102; July 18–23, 2010; Vienna, Austria.
49. Garcia F, Climent N, Assoumou L, et al.. A therapeutic dendritic cell-based vaccine for HIV-1 infection. J Infect Dis. 2011;203:473–478.
50. Kloverpris H, Karlsson I, Bonde J, et al.. Induction of novel CD8+ T-cell responses during chronic untreated HIV-1 infection by immunization with subdominant cytotoxic T-lymphocyte epitopes. AIDS. 2009;23:1329–1340.
51. Van Gulck E, Vlieghe E, Vekemans M, et al.. mRNA-based dendritic cell vaccination induces potent antiviral T-cell responses in HIV-1-infected patients. AIDS. 2012;26:F1–F12.
52. Jacobson JM, Pat Bucy R, Spritzler J, et al.. Evidence that intermittent structured treatment interruption, but not immunization with ALVAC-HIV vCP1452, promotes host control of HIV replication: the results of AIDS Clinical Trials Group 5068. J Infect Dis. 2006;194:623–632.
53. Shan L, Deng K, Shroff NS, et al.. Stimulation of HIV-1-specific cytolytic T lymphocytes facilitates elimination of latent viral reservoir after virus reactivation. Immunity. 2012;36:491–501.
54. Ehl S, Barchet W, Oehen S, et al.. Donor cell persistence and activation-induced unresponsiveness of peripheral CD8+ T cells. Eur J Immunol. 2000;30:883–891.
55. Todryk SM, Pathan AA, Keating S, et al.. The relationship between human effector and memory T cells measured by ex vivo and cultured ELISPOT following recent and distal priming. Immunology. 2009;128:83–91.
56. Keating SM, Bejon P, Berthoud T, et al.. Durable human memory T cells quantifiable by cultured enzyme-linked immunospot assays are induced by heterologous prime boost immunization and correlate with protection against malaria. J Immunol. 2005;175:5675–5680.
57. Ndhlovu ZM, Proudfoot J, Cesa K, et al.. Elite controllers with low to absent effector CD8+ T cell responses maintain highly functional, broadly directed central memory responses. J Virol. 2012;86:Epub ahead of print.
58. Friedrich D, Jalbert E, Dinges WL, et al.. Vaccine-induced HIV-specific CD8+ T cells utilize preferential HLA alleles and target-specific regions of HIV-1. J Acquir Immune Defic Syndr. 2011;58:248–252.