Clinical trial design and the demographics of trial participants
The original clinical trial plan included the enrollment of a total of 120 healthy, HIV-uninfected adult subjects between ages of 18 and 50 years. They were randomized into a two-part dose-escalation and regimen comparison clinical trial. Twenty-four participants were enrolled in Part A, the dose-escalation study for the protein vaccine EP-1043. Each of groups 1 and 2 had 12 participants that were randomized to receive vaccine or control in a (10:2) ratio. In Part B, extended safety and immunogenicity were evaluated for the combination of a protein vaccine EP-1043 and a DNA vaccine EP-1090, as well as each vaccine alone. For groups in Part B, 96 participants were to be concurrently randomized to groups 3, 4 and 5: 24 participants to be randomized to group 3 according to a (vaccine:placebo)=(20:4) ratio, and 36 participants to be randomized to each of groups 4 and 5 according to a (vaccine:placebo)=(30:6) ratio (). Although a sufficient amount of the protein vaccine was made and vialed for the clinical trial, there was an incomplete documentation of the sterilization of the vaccine vials and thus the vials were recalled. Part B was not executed as originally planned. Only 84 of planned 120 volunteers were enrolled into the actual clinical trial, 70 received vaccines, and 14 received placebo controls (). Thus, safety and immunogenicity results on these 84 subjects are reported. We belive this problem will not likely to affect the interprestion of safety and immunogenicity data obtained from this clinical trial, nor will it alter significantly the vaccine’s potential utility for future clincial trials.
Study volunteers were enrolled from three US cities: Rochester, New York; Baltimore, Maryland; and San Francisco, California. About 2/3 of the trial participants were male (55/84), and 1/3 were female (29/84). Half of them were white, non-Hispanic (49/84), approximately 1/5 were black (17/84), followed by a small proportion of Hispanic (5/84), and Asian/Other participants (13/84). The median age of these volunteers was 35 years. The overwhelming majority of them were between 21–50 (80/84) years of age, and the rest were between the ages of 18 to 20 (4/84).
The EP-1043 vaccine is safe and well-tolerated
The vaccines were safe and well tolerated when either used alone or in combination. A total of 285 adverse events (AEs) were reported by 83% (71/84) of trial participants. The majority of them reported either mild (30/70), or moderate AEs (29/70); a small proportion of subjects suffered severe AEs (10/70). Two participants reported life-threatening events: one was hospitalized with gastric ulcer bleeding 73 days after the 3rd vaccination of the EP-1090 vaccine, and another had a significant elevation of CPK levels noted at the final visit (10 months after the last vaccination with 0.2mg of the EP-1043 vaccine). The episode of elevated CPK was considered likely due to recent weight training exercises. Neither of these events was considered related to the use of the vaccine product. Other reports of severe AEs include urinary tract infection, influenza infection, low back pain, pharyngitis, suicide attempt, acute gastroenteritis, and exacerbation of pre-existing diverticulitis (). However, none of the severe AEs were related to the vaccine products. Most commonly reported reactogenicity symptoms were local pain and tenderness at the site of injection; systemic symptoms related to vaccine products were malaise/fatigue, myalgia, arthragia, headache, nausea, vomiting, and fever/chills ().
The EP-1043 vaccine elicits potent CD4+ T cell responses
Immunogencity assessment was performed on all trial participants with PBMC samples obtained 2 weeks after the 2nd vaccination (Day 42), and some subjects after the 4th vaccination (Day 182). shows the sum of CD4+ T cell responses to HIV antigens above background levels for either or both cytokines (IFNγ+IL2+, IFNγ+IL2-and IFNγ-IL2+). The combined response (IL2 and/or IFNγ) is defined as the sum of the subset responses after stimulation with a pool of HTL peptides included in the EP-1043 vaccine. Participants with a positive combined cytokine response were included in the plots. The ICS assay was performed on 22 samples from 12 control subjects (from days 42 and 182), and on samples from 67 and 58 vaccine recipients at day 42 (2 weeks after the 2nd vaccination), and day 182 (2 weeks after the 4th vaccination), respectively. After 2 vaccinations (Day 42), none of the control recipients (C) had a positive response. In subjects enrolled in groups receiving the protein vaccine (T1, T2, T3 and T5), 68% (32/47) of subjects had a positive CD4+ T response, and the magnitudes of IL-2- or IFNγ-producing CD4+ T cells were as high as 0.1–0.5% of total CD4+ T cells. The levels of these responses, however, were not boosted by subsequent vaccinations, and the proportion of positive responders remained at about 70% (27/39) (Day 182). Since CD8+ T cell responses were not induced by the EP-1090 vaccine either alone or in combination with the EP1043 vaccine (groups T4 and T5), only CD4+ T cell responses elicited by the EP-1043 vaccine were further examined.
CD4+ T cells producing IFN-γ or IL-2 are elicited by EP-1043 vaccine
The EP-1043 vaccine elicits poly-functional CD4+ T cell responses
In a murine model of leishmaniasis, poly-functional CD4 T cells have been shown to be a key effector for protection [10
]. In human HIV infections, these cells are hypothesized to be more important for protection than those that only produce a narrow range of cytokines [11
]. We next examined whether EP-1043 could elicit poly-functional T cells using ICS assay for TNF-α and IL-4 in additional to IFN-γ and IL-2. shows an example of the flow cytometric staining profile for the CD4+ T cell response in one participant. In this individual, the most abundant vaccine-induced cytokine made by CD4 cells was IL-2, and of these IL-2-producing cells, 63% produce TNF-α and a smaller proportion produce IFN-γ or IL-4.
Flow cytometric profile of cytokine-secreting CD4+ T cells
summarizes the poly-functionality of the vaccine-induced CD4+ T cells for 27 trial participants who received the HTL vaccine alone or in combination with the DNA vaccine at 2 weeks after the 4th vaccination (Day 182). The degree of poly-functionality is similar for the two protein doses and when DNA is co-administered. Most cells produced either one or two cytokines (40% and 45%, respectively), approximately 10% produced three, and none produced all four cytokines measured (). To determine which cytokine or cytokine combinations were produced, cells with each type of poly-functionality were further examined. Among cells producing one cytokine, IL-2 was most commonly produced; TNF-α or IL-4 was produced by a smaller percentage of cells; none produced IFN-γ only. Among cells producing two cytokines, IL-2 in combination with TNF-α was the dominant combination, followed by the IL-2 and IL-4 combination. Among cells producing three cytokines, only two of the four potential combinations were observed. Both included IL-2 and TNF-α, and were produced in combination with either IL-4 or IFN-γ ().
Cytokine-secreting CD4+ T cells are induced by EP-1043 vaccine