Conceived and designed the experiments: EN AG RE GT. Performed the experiments: EN AG RE GO CO JC. Analyzed the data: EN AG RE GO CO JC. Contributed reagents/materials/analysis tools: EN GT. Wrote the paper: EN AG RE.
Current address: Division of Infectious Diseases, Department of Medicine, University of California Los Angeles, Los Angeles, California, United States of America,
Current address: Regeneron Pharmaceuticals, Inc., Tarrytown, New York, United States of America
Current address: Department of Pathology, University of Massachusetts School of Medicine, Worcester, Massachusetts, United States of America
The authors have declared that no competing interests exist.
The objectives of this non-randomized, non-blinded, dose-escalating Phase I clinical trial were to assess the safety, reactogenicity and immunogenicity of ICC-1132 formulated with Alhydrogel (aluminum hydroxide) in 51 healthy, malaria-naive adults aged 18 to 45 years. ICC-1132 (Malariavax) is a recombinant, virus-like particle malaria vaccine comprised of hepatitis core antigen engineered to express the central repeat regions from
ClinicalTrials.gov
An effective vaccine is needed to prevent or attenuate disease from
Virus-like particles have been used recently as highly immunogenic delivery platforms for a variety of vaccines
Circumsporozoite protein is comprised of a central portion of amino acid repeats (NANP) representing dominant T cell-dependent B cell epitopes
After initiation of this present study in the USA, the ICC-1132 vaccine was tested in other, more limited studies in Europe, the results of which have been published
The protocol for this trial and supporting CONSORT checklist are available as supporting information; see
The trial took place on the campuses of the University of Maryland at Baltimore and at College Park. The study methods and rationale, along with the study consent was explained to potential healthy, adult participants aged 18—45. A written exam was administered to potential participants to assess their understanding of the study procedures, rationale and expected outcomes. Consented participants were screened by medical history, physical examination and laboratory analysis of hematologic and serologic markers. Exclusion criteria included medical history or serologic indications of malaria, HIV, HBsAg or any significant cardiovascular, hepatic or renal function abnormalities.
The vaccine has been described
The T* epitope, EYLNKIQNSLSTEWSPCSVT, is inserted starting at amino acid V149. The amino terminus is noted as H2N and the starting amino acid is labelled as M1. Adapted from Bottcher,
ICC-1132 was adsorbed to aluminum hydroxide (Alhydrogel; Superfos, Frederikssund, Denmark) with >95% adsorption as determined by measurement of residual unbound protein. Each 1 mL of vaccine contained 1 mg of aluminum as Al(OH)3. The saline formulation contained the same concentration of ICC-1132, but did not contain Alhydrogel. The vaccine was produced, purified and formulated by Apovia, Inc.
The study was originally designed as a double-masked, dose-escalating trial comparing the three dose levels (10, 20 and 50 mcg) of ICC-1132 in saline to the same three dose levels of ICC-1132 adsorbed to Alhydrogel. In January 2003 the trial was delayed because the saline formulation of the vaccine was found to be unstable after six to nine months' storage at 4°C. The saline formulation was subsequently removed from the study, leaving the stable alum preparation to be tested. The study was redesigned to assess the 20 and 50 mcg doses of the Alhydrogel vaccine in a non-masked, dose escalating fashion. The study was conducted according to GCP guidelines. Ethical approval for the study was obtained from the Institutional Review Boards of the University of Maryland, Baltimore and University of Maryland, College Park and the New York University School of Medicine.
The primary objectives were (1) to compare the safety and reactogenicity of ICC-1132 in saline and with the Alhydrogel formulation in healthy, malaria-naive human adults, and (2) to assess the immunogenicity of the two ICC-1132 vaccine formulations. A secondary objective was to vaccinate a sufficient number of volunteers who would agree to participate in a concurrent malaria challenge trial. The immunogenicity data for the saline formulation is abbreviated for reasons described. The malaria challenge trial was cancelled because of lack of sufficient immunogenicity, predefined as a dose cohort median IFA of ≥1500.
Local and systemic reactions were assessed for 30 minutes following each injection. Clinical assessments were carried out at 1, 2, 7, 14, 28 and 56 days after each injection, at day 84 after the second injection, and day 168 after the third injection. Telephone interviews were conducted at 4 days after each injection and volunteers maintained a daily diary to collect adverse events and twice daily body temperature recordings in the seven days immediately after each injection.
Tenderness, pain, erythema, induration and pruritus, at the site of injection, were graded.
Solicited systemic variables evaluated included fever, chills, malaise, headache, photophobia, anorexia, nausea, vomiting, abdominal pain, myalgia, arthralgia and rash. Non-solicited adverse event reported by a volunteer was recorded and an assessment of causality was performed by the investigators.
Local and systemic adverse events were graded according to the following schema: Grade 1 mild, no change in activity and/or no medication necessary; Grade 2 moderate, requires change in activity and/or medication; Grade 3 severe, bed rest required/inability to perform normal activities and/or medical intervention other than medication alone (such as an outpatient visit in emergency department or clinic, excluding hospitalization).
Urinalysis, hematological and biochemical safety analysis was carried out prior to vaccine injections, 2, 14, 28 days after all injections, 84 days after the second injection, and 56 and 168 days after the third injection.
Serum samples for serological assays and peripheral blood mononuclear cells (PBMC) for cellular immune assays were obtained at the time of each immunization, 14 and 28 days after each injection, 84 days after the second injection, and 56 and 168 days after the third injection. Methods used for ELISA, IFA and CSP reactions are identical to those previously published
The PBMC were Ficoll purified from blood collected in citrate buffer Vacutainer (BD Biosciences, San Diego, CA). Short term TCL were used in the assays by expanding PBMC (2x106/mL) with a single
In this phase 1 descriptive study focusing on safety and immunogenicity, sample sizes were derived from logistic considerations, rather than by power analyses. The larger number of volunteers in the 50 mcg cohort was designed to yield sufficient interested volunteers to enable a malaria challenge trial after the third vaccination.
Using SAS, a randomization list was prepared in advance of vaccination activities with randomized block of
Assignments to vaccine groups from this randomization were placed in individual sealed envelopes and were provided to the immunization team. Randomization assignment was masked from investigators assessing post-injection adverse events.
The original study statistician generated the allocation sequence. Subjects in the 10 mcg cohort and the first three subjects in the 20 mcg cohort were randomized by computer to receive ICC-1132 in saline or ICC-1132 adsorbed to Alhydrogel. All 32 subsequent subjects received ICC-1132 adsorbed to Alhydrogel, and were allocation to either the 20 mcg or 50 mcg cohorts on a first come, first serve basis, such that the first eight available subjects were assigned the 20 mcg cohort, while the last 24 subjects were assigned the 50 mcg cohort.
The participants, vaccine administrators and clinical staff providing safety follow-up were blinded to which formulation of vaccine subjects had received prior to removal of the saline formulated vaccine from the study. Neither the subjects nor investigators were blinded once the saline formulation was removed from the trial, because only one formulation was used and because the dose-escalation design required that the safety of the 20 mcg dose be evaluated prior to administration of the 50 mcg dose.
Quantitative data were assessed for normal distribution and log-transformation was performed where appropriate. Differences among proportions were compared using Fisher's exact tests, differences between medians were compared using the Wilcoxon Rank Sum test and Spearman's Rank Correlation test was used to assess association strengths. All tests were two sided. A
A total of 51 volunteers received at least one injection and 29 volunteers completed the study to receive all three injections of ICC-1132 adjuvanted to Alhydrogel (
A total of 51 volunteers received at least one injection and 29 volunteers completed the study to receive all three injections of ICC-1132 adjuvanted to Alhydrogel.
The distribution of volunteers by gender and ethnicity was similar statistically amongst all dose and formulation groups (
The vaccine was well tolerated at the dose levels examined in this study. No severe (grade 3) adverse events occurred, nor any clinically significant laboratory abnormalities attributable to vaccination. One serious adverse event occurred, unrelated to vaccination, in a volunteer who became pregnant within three months of receiving the third 20 mcg injection. She delivered a healthy infant at 40 weeks. No significant differences in reactogenicity were noted between dose cohorts, between the first, second and third vaccination, nor between Alhydrogel and saline vaccine groups.
Of 125 total vaccinations administered to 51 volunteers, local adverse events were principally limited to mild tenderness or pain at the injection site in 46 volunteers. In addition, four volunteers had local pruritus and one volunteer each had an episode of induration and erythema (
Local | |||||
Tenderness | Pain | Pruritus | Induration | Erythema | |
Grade 1 | 68 | 53 | 4 | 1 | 1 |
Grade 2 | 1 | 9 | 0 | 0 | 0 |
Grade 3 | 0 | 0 | 0 | 0 | 0 |
Total | 69 | 62 | 4 | 1 | 1 |
Grade 1: Mild; no change in activity and/or no medication necessary
Grade 2: Moderate; requires change in activity and/or medication
Grade 3: Severe; bed rest required and/or medical intervention other than medication alone (such as an outpatient visit in emergency department or clinic, excluding hospitalization)
Headache and myalgia were the most common manifestations of systemic reactogenicity, followed by other elicited symptoms (
Systemic | ||||||
HA | Myalgia | Subjective Fever | Nausea | Arthralgia | Anorexia | |
Grade 1 | 14 | 12 | 4 | 3 | 4 | 4 |
Grade 2 | 6 | 3 | 1 | 3 | 0 | 0 |
Grade 3 | 0 | 0 | 0 | 0 | 0 | 0 |
Total | 20 | 15 | 5 | 6 | 4 | 4 |
HA = headache
One episode of temperature to 37.8 C
Depending upon the vaccine dose, 95—100% of volunteers developed specific responses to the ICC-1132 vaccine immunogen (
Antigen | ICC-1132 | HBc | (T1B)4 | IFA | ||||||||
Post Dose | 1 | 2 | 3 | 1 | 2 | 3 | 1 | 2 | 3 | 1 | 2 | 3 |
10 µg | 320 | 16255 | NA | 63 | 4561 | NA | 67 | 269 | NA | NA | 226 | NA |
(88) | (100) | - | (33) | (100) | - | (25) | (75) | - | - | (63) | - | |
20 µg | 123 | 905 | 3121 | 73 | 905 | 2319 | <80 | 106 | 101 | NA | 80 | 66 |
(50) | (100) | (100) | (50) | (100) | (100) | (13) | (50) | (43) | - | (25) | (29) | |
50 µg | 88 | 1004 | 2661 | 49 | 577 | 2463 | 45 | 63 | 138 | NA | 57 | 118 |
(48) | (95) | (94) | (17) | (90) | (94) | (8) | (38) | (53) | - | (25) | (53) |
ELISA
Indirect immunofluorescence assay
Differences between 10, 20 and 50 µg cohort percent responders not statistically significant
Vaccination on days 0, 56 and 168
NA = Not available
The anti-ICC-1132 and anti-HBc GMTs were significantly higher in the 10 mcg cohort compared to the 20 and 50 mcg cohorts after the second injection, although the percent responders to the two antigens (90—100%) were nearly identical in the three cohorts (
Malaria specific anti-(T1B)4 response was significantly higher in the 10 mcg cohort compared to the 50 mcg cohort after the second injection (
Anti-ICC-1132, anti-HBc and anti-(T1B)4 IgG1 and IgG3 subtypes, typical of TH1-type immune responses, developed preferentially over IgG2 and IgG4 subtypes (
Two (volunteers 1 and 10) of the seven volunteers in the 10 mcg alum cohort (those with the most robust malaria specific humoral responses) developed positive CSP reactions, which demonstrates the presence of specific antibodies capable of cross-linking surface CS protein on the viable sporozoite. Inhibition in the TSNA using
As measured by IFA against whole sporozoite (gradient bars), (T1B)4 ELISA (hatched bars) and transgenic sporozoite neutralization assay (TSNA) (percentages above bars). For the TSNA, 94.5% inhibition was obtained using a positive control monoclonal antibody specific for
Depending on vaccine dose, 65—75% of the volunteers' demonstrated T cell proliferation in response to ICC-1132 and 47—75% had proliferation to rCS. The magnitude of responses to these two antigens was similar (
Cohort | Day 0 | Day 84 | Day 196 | |||
ICC-1132 | rCS | ICC-1132 | rCS | ICC-1132 | rCS | |
10 µg | 3033 | 4459 | 21744 | 23496 | NA | NA |
- | - | (75) | (75) | - | - | |
20 µg | 587 | 3484 | 15903 | 4736 | 10451 | 13772 |
- | - | (67) | (75) | (57) | (29) | |
50 µg | 310 | 462 | 3636 | 11948 | 10035 | 11063 |
- | - | (13) | (29) | (65) | (47) |
Data from 4 volunteers
Data from 7 volunteers
Responses 14 days after the second dose of ICC-1132 (Day 70)
Data from 8 volunteers
Data from 14 volunteers
Data from 17 volunteers
Differences between 10, 20 and 50 µg cohorts
Vaccination on days 0, 56 and 168
NA = Not available
Cohort | Antigen | ||
rCS NF-54 | (T | (T1B)4 | |
10 µg | 12114 (0/4) | 47 (1/4) | 52 (0/4) |
20 µg | 1515 (1/7) | 268 (2/7) | 242 (1/7) |
50 µg | 2831 (7/17) | 147 (4/17) | 55 (0/17) |
Total Percent Responders | 29 (8/28) | 25 (7/28) | 4 (1/28) |
T universal epitope
Responses 14 days after the second dose of ICC 1132 (Day 70)
Responses 28 days after the third dose of ICC 1132 (Day 196)
Differences between cohorts not statistically significant
This
Our most intriguing finding is the difference between the 10 and 50 mcg cohorts in terms of humoral immunogenicity of the vaccine. For all antigens evaluated, the 10 mcg cohort response was superior to that of the 50 mcg after the second injection. Intercohort differences may have been an effect of the manner in which antigen bound to the adjuvant in the lower versus higher dose vaccines. Although unusual, it is not without precedent that a lower immunogen dose would lead to a more robust humoral response
Volunteers with preexisting anti-HBc titers (two volunteers in the 10 alum and one volunteer in the 50 mcg cohort) demonstrated more robust humoral responses to immunogen and HBc, with titers consistently higher than their respective cohorts' GMT. There was not a noticeably increased response in these individuals against malaria-specific antigens. This suggests that, in these previously anti-HBc positive individuals, the more robust response against the immunogen was specific for HBc epitopes. Indeed, the higher antibody titers seen against both the immunogen and HBc in all the volunteers could imply that the targeted epitopes in the immunogen are shared HBc epitopes, rather than malaria epitopes. The proliferation data shows that the cellular immune response is targeting malaria-specific responses, as the median δCPM against rCS and ICC-1132 are similar at day 196 (
Volunteers developed TH1-type antibody, with anti-CS IgG1 and IgG3 subtypes developing preferentially over IgG2 and IgG4, as found in previous study of this vaccine
The good correlation between (T1B)4 ELISA and IFA titers suggests that anti-repeat antibodies elicited by the ICC-1132 vaccine recognize native CS on the sporozoite (
After initiation of this present study in the USA, the vaccine was tested in other, more limited studies in Europe, the results of which have been published
In summary, the candidate vaccine, ICC-1132, was safe and well-tolerated at all dose levels examined in this trial. ICC-1132 was poorly immunogenic when adjuvanted with alum. The immunogenicity of the candidate vaccine may be improved through combination with a more potent adjuvant.
CONSORT Checklist
(0.10 MB PDF)
Final Trial Protocol
(0.78 MB PDF)
The authors would like to thank the volunteers for their participation in this trial. We also thank the nursing and staff of the Center for Vaccine Development College Park and Baltimore Units (particularly Joanna Becker and Karen Ball), the University of Maryland's General Clinical Research Center, the University of Maryland's Investigational Drug Service, and acknowledge the expert technical assistance of Diana Barrios Rodriguez, Rita Altszuler and Lissette Schettini. Thanks as well to Pramod Sarpotdar and Ashley Birkett of Apovia, Inc. for the production and formulation of ICC-1132. IL-2 was provided by NIH AIDS repository.