Safety and Immunogenicity of an Intramuscular Helicobacter pylori Vaccine in Noninfected Volunteers: A Phase I Study

Introduction: Helicobacter pylori infection is among the most common human infections and the major risk factor for peptic disease and gastric cancer. Immunization with vaccines containing the H pylori vacuolating cytotoxin A (VacA), cytotoxin-associated antigen (CagA), and neutrophil-activating protein (NAP), alone or in combination, have been shown to prevent experimental infection in animals. Aim: We sought to study the safety and immunogenicity of a vaccine consisting of recombinant VacA, CagA, and NAP given intramuscularly with aluminium hydroxide as an adjuvant to noninfected healthy subjects. Methods: This controlled, single-blind Phase I study randomized 57 H pylori-negative volunteers into 7 study arms exploring 2 dosages (10 and 25 μg) of each antigen and 3 schedules (0, 1, 2 weeks; 0, 1, 2 months; and 0, 1, 4 months) versus alum controls. All participants were followed for 5 months. Thirty-six subjects received a booster vaccination 18–24 months after the completion of the primary vaccination. Results: Local and systemic adverse reactions were mild and similar in placebo and vaccine recipients on the monthly schedules. All subjects responded to 1 or 2 of the antigens and 86% of all vaccines mounted immunoglobulin G antibody responses to all 3 antigens. Vaccinees exhibited an antigen-specific cellular response. Vaccination 18–24 months later elicited anamnestic antibody and cellular responses. Conclusions: This intramuscular H pylori vaccine demonstrated satisfactory safety and immunogenicity, produced antigen-specific T-cell memory, and, therefore, warrants further clinical study. Introduction: Helicobacter pylori infection is among the most common human infections and the major risk factor for peptic disease and gastric cancer. Immunization with vaccines containing the H pylori vacuolating cytotoxin A (VacA), cytotoxin-associated antigen (CagA), and neutrophil-activating protein (NAP), alone or in combination, have been shown to prevent experimental infection in animals. Aim: We sought to study the safety and immunogenicity of a vaccine consisting of recombinant VacA, CagA, and NAP given intramuscularly with aluminium hydroxide as an adjuvant to noninfected healthy subjects. Methods: This controlled, single-blind Phase I study randomized 57 H pylori-negative volunteers into 7 study arms exploring 2 dosages (10 and 25 μg) of each antigen and 3 schedules (0, 1, 2 weeks; 0, 1, 2 months; and 0, 1, 4 months) versus alum controls. All participants were followed for 5 months. Thirty-six subjects received a booster vaccination 18–24 months after the completion of the primary vaccination. Results: Local and systemic adverse reactions were mild and similar in placebo and vaccine recipients on the monthly schedules. All subjects responded to 1 or 2 of the antigens and 86% of all vaccines mounted immunoglobulin G antibody responses to all 3 antigens. Vaccinees exhibited an antigen-specific cellular response. Vaccination 18–24 months later elicited anamnestic antibody and cellular responses. Conclusions: This intramuscular H pylori vaccine demonstrated satisfactory safety and immunogenicity, produced antigen-specific T-cell memory, and, therefore, warrants further clinical study. Helicobacter pylori, a gram-negative bacterium colonizes the human gastric mucosa. It is among the most common chronic bacterial infections in humans1Covacci A. Telford J. Del Giudice G. et al.Helicobacter pylori, virulence and genetic geography.Science. 1999; 284: 1328-1333Crossref PubMed Scopus (963) Google Scholar and affects approximately half the world's population. Its prevalence in Western countries ranges from 10% to 60%2Graham D.Y. Malaty H.M. 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Codolo G. et al.The neutrophil-activating protein of Helicobacter pylori promotes Th1 immune responses.J Clin Invest. 2006; 116: 1092-1101Crossref PubMed Scopus (258) Google Scholar The results of prophylactic vaccination in animal models have been encouraging.14Del Giudice G. Covacci A. Telford J. et al.The design of vaccines against Helicobacter pylori and their development.Annu Rev Immunol. 2001; 19: 523-563Crossref PubMed Scopus (201) Google Scholar, 15Del Giudice G. Michetti P. Inflammation, immunity and vaccines for Helicobacter pylori.Helicobacter. 2004; 9: 23-28Crossref PubMed Scopus (29) Google Scholar Immunization with recombinant VacA, CagA, and NAP, singly or in combination, together with mucosal adjuvants, confers protection in mice.30Marchetti M. Arico B. Burroni D. et al.Development of a mouse model of Helicobacter pylori infection that mimics human disease.Science. 1995; 267: 1655-1658Crossref PubMed Scopus (538) Google Scholar, 31Marchetti M. Rossi M. Giannelli V. et al.Protection against Helicobacter pylori infection in mice by intragastric vaccination with H. pylori antigens is achieved using a non-toxic mutant of E. coli heat-labile enterotoxin (LT) as adjuvant.Vaccine. 1998; 16: 33-37Crossref PubMed Scopus (163) Google Scholar, 32Satin B. Del Giudice G. Della Bianca S. et al.The neutrophil-activating protein (HP-NAP) of Helicobacter pylori is a protective antigen and a major virulence factor.J Exp Med. 2000; 191: 1567-1576Crossref Scopus (275) Google Scholar These antigens were further investigated in a Beagle dog model.33Rossi G. Rossi M. Vitali C.G. et al.A conventional Beagle dog model for acute and chronic infection with Helicobacter pylori.Infect Immun. 1999; 67: 3112-3120PubMed Google Scholar, 34Rossi G. Fortuna D. Pancotto L. et al.Immunohistochemical study of lymphocyte populations infiltrating the gastric mucosa of Beagle dogs experimentally infected with Helicobacter pylori.Infect Immun. 2000; 68: 4769-4772Crossref PubMed Scopus (28) Google Scholar VacA, CagA, and NAP given intramuscularly (IM) with aluminium hydroxide as adjuvant protected dogs against a challenge with H pylori (Del Giudice et al., unpublished data) or eradicated experimental infection.35Rossi G. Ruggiero P. Peppoloni S. et al.Therapeutic vaccination against Helicobacter pylori in the beagle dog experimental model: safety, immunogenicity, and efficacy.Infect Immun. 2004; 72: 3252-3259Crossref PubMed Scopus (86) Google Scholar Other groups have also shown efficacy of parenteral vaccination in mice using whole-cell bacterial preparations in combination with aluminium hydroxide.36Gottwein J.M. Blanchard T.G. Targoni O.S. et al.Protective anti-Helicobacter immunity is induced with aluminium hydroxide or complete Freund's adjuvant by systemic immunization.J Infect Dis. 2001; 184: 308-314Crossref PubMed Scopus (87) Google Scholar, 37Eisenberg J.C. Czinn S.J. Garhart C.A. et al.Protective efficacy of anti-Helicobacter pylori immunity following systemic immunization of neonatal mice.Infect Immun. 2003; 71: 1820-1827Crossref PubMed Scopus (40) Google Scholar The purpose of this Phase I study was to assess the safety and immunogenicity of VacA, CagA, and NAP, with an aluminium hydroxide adjuvant, in healthy, H pylori-negative subjects. The vaccine (HP3) consisted of sterile purified recombinant VacA, CagA, and NAP adsorbed onto aluminium hydroxide (1 mg/mL), in 0.5 of isotonic buffer contained in prefilled syringes for IM injection into the deltoid muscle. Antigens were expressed in E coli as full-length proteins and exhibited >95% purity after appropriate chromatographic steps as previously described.32Satin B. Del Giudice G. Della Bianca S. et al.The neutrophil-activating protein (HP-NAP) of Helicobacter pylori is a protective antigen and a major virulence factor.J Exp Med. 2000; 191: 1567-1576Crossref Scopus (275) Google Scholar, 38Covacci A. Censini S. Bugnoli M. et al.Molecular characterization of the 128-kDa immunodominant antigen of Helicobacter pylori associated with cytotoxicity and duodenal ulcer.Proc Natl Acad Sci U S A. 1993; 90: 5791-5795Crossref PubMed Scopus (1129) Google Scholar, 39Manetti R. Massari P. Burroni D. et al.Helicobacter pylori cytotoxin: importance of native conformation for induction of neutralizing antibodies.Infect Immun. 1995; 63: 4476-4480Crossref PubMed Google Scholar Formulations containing low (10 μg) or high (25 μg) doses of the 3 antigens were tested. The placebo contained 0.5 mg of aluminium hydroxide alone. All volunteers gave informed consent to participate. Exclusion criteria included a history of, treatment for, or other evidence of H pylori infection and treatment within the past 4 weeks with antibiotics, bismuth, or proton pump inhibitors. Subjects were serologically negative for H pylori at screening (Enzygnost, Anti-Helicobacter pylori II: Dade Behring, Marburg, Germany) and underwent a urea breath test (13Rupnow M.F. Shachter R.D. Owens D.K. et al.Quantifying the population impact of a prophylactic Helicobacter pylori vaccine.Vaccine. 2001; 20: 879-885Crossref PubMed Scopus (48) Google ScholarC-UBT; Pylobactell, Torbet Laboratories Ltd, Norwich, UK) at screening, the final visit of the primary series, and before the administration of the booster vaccination; all tests were negative. This phase I, randomized, controlled, single-blind, dose-ranging, and schedule-finding study was carried out under a US Investigational Drug Application and received approval from the Medical Ethics Committees of the University of Magdeburg and the Humboldt University, Berlin, Germany. Patients were blinded to their treatment group assignment (single blind), as were personnel involved in running the immunogenicity assays. Fifty-seven healthy adult (aged 18–40) volunteers meeting all entry criteria (including use of appropriate contraception for women) were enrolled in 2001 and randomly assigned to 1 of 7 groups (Table 1). Thirty-eight volunteers (18 female; mean age, 29.9 ± 6.3 years) received the HP3 vaccine and/or placebo on 1 of 2 monthly schedules, and 19 (12 female; mean age, 28.9 ± 5.7 years) received the vaccine on the weekly schedule. All but 1 of the 57 volunteers received all scheduled injections and all were included in the analyses. The weekly regimen was included to assess safety and immunogenicity of a schedule compatible with the current 2-week treatment for chronic infection.Table 1Treatment SchedulesGroup no.No. of volunteers per groupMonth 0Month 1Month 2Month 4No. of volunteers per groupMonths 18–24Primary series: Monthly schedule (n = 38)Booster vaccination (n = 21) 17Low dose⁎Low dose = 10 μg of each antigen.Low doseLow dosePlacebo5Low dose⁎Low dose = 10 μg of each antigen. 27High dose‡High dose = 25 μg of each antigen.High doseHigh dosePlacebo5High dose‡High dose = 25 μg of each antigen. 37Low doseLow dosePlaceboLow dose5Low dose 48High doseHigh dosePlaceboHigh dose6High dose 59ControlControlControlControl—§No alum control given in those receiving the booster dose 18–24 months after completion of the primary series.—Week 0Week 1Week 2Weekly schedule (n = 19)n = 15 69Low doseLow doseLow dose7Low dose 710High doseHigh doseHigh dose8High dose Low dose = 10 μg of each antigen.‡ High dose = 25 μg of each antigen.§ No alum control given in those receiving the booster dose 18–24 months after completion of the primary series. Open table in a new tab To investigate persistence and boostability of the vaccine-induced immune responses, 18–24 months later 36 subjects received a fourth dose of the HP3 vaccine, either at low or high dose, based on the group to which they belonged. Blood samples were taken before and 1 month after each injection (groups 1, 2, 3, 4, and 5), or before each injection and at month 5 (groups 6 and 7). Additional blood samples were taken before and 4 weeks after the booster vaccination. Volunteers were observed for 30 minutes after each injection to detect immediate local or systemic reactions or other adverse events. The subjects received diary cards to record injection site (pain, erythema, or induration) and/or systemic (fatigue, malaise, fever, chills, myalgia, arthralgia, nausea, headache, and rash) reactions as well as body temperature daily for 6 days postinjection. Serious adverse events were defined as events that were fatal or life threatening, caused hospitalization, resulted in significant, persistent, or permanent disability, or required intervention to prevent permanent impairment or damage. All adverse events and serious adverse events were recorded throughout the study, as were any medications. All subjects were followed for 5 months after their first injection and had follow-up visits 1 month after their third injection with additional follow-up visits at 18–24 months. Ninety-six-well, flat-bottomed microtiter plates (Nunc, Roskilde, Denmark) were coated with 1 of the recombinant proteins in 100 μL phosphate-buffered saline (PBS), pH 7.4. Full-length CagA (lot 010101-CAG07/28) was used at 0.9 μg/mL and coating occurred over 1.5 hours at 30°C. NAP (lot RS020101) was used at 1 μg/mL, and coating lasted 2.5 hours at 30°C. VacA (lot RS030401) was used at 2 μg/mL with coating for 1 hour at room temperature (RT). After 3 washes with PBS containing 0.05% (v/v) Tween-20, the plates were incubated with 250 μL/well of a solution of polyvinylpyrrolidone-15 (Serva, Heidelberg, Germany) for 1.5 hours at RT. After further washes, serial doubling dilutions of serum samples (starting from 1:400), diluted in PBS Tween-20 containing 2% (w/v) bovine serum albumin (BSA; Sigma Chemical Co, St Louis, MO) were added to the plates. Antibodies were allowed to react for 1 hour at 37°C (CagA enzyme-linked immunosorbent assay [ELISA]) or 30°C (NAP and VacA ELISA). Bound immunoglobulin (Ig)G antibodies were quantified by alkaline phosphatase-conjugated polyclonal affinity-purified goat anti-human IgG (Sigma Chemical Co), diluted 1:2000 in PBS–Tween-20–BSA and incubated for 3 hours at RT. After washing, 100 μL of p-nitrophenylphosphate (Sigma Chemical Co) at 1 mg/mL in diethanolamine buffer were added to each well. The chromogenic reaction proceeded for 40 minutes (CagA and VacA ELISA) or 1 hour (NAP ELISA) in the dark at RT and was stopped with 4 N NaOH. Optical densities were read at 405 and 650 nm. Antigen-specific IgG antibody titers were expressed as ELISA units (EU) and determined by interpolation relative to a standard curve constructed by a serial dilution of a standard positive control. Cut-off values of 20, 70, and 100 EU were determined for the CagA, NAP, and VacA ELISA, respectively, using serum samples from individuals negative for H pylori infection. Seroconversion was defined as EU values below the cut-off levels before vaccination that became positive after vaccination, or when EU values above cut-off levels before vaccination increased ≥3-fold after vaccination. Peripheral blood mononuclear cells (PBMC) isolated by Ficoll gradient centrifugation were stimulated in triplicate in 96-well-round-bottomed plates at 1 × 105 cells/well with recombinant CagA, VacA, or NAP at 5 μg/mL for 3 days. Proliferative response was assayed as incorporation of 3Frenck Jr, R.W. Clemens J. Helicobacter in the developing world.Microbes Infect. 2003; 5: 705-713Crossref PubMed Scopus (245) Google ScholarH-thymidine during the last 18 hours of incubation. Results were expressed as stimulation index, namely, the ratio between the counts obtained in wells stimulated with antigens and those obtained in control wells receiving medium alone. Phytohaemagglutinin and tetanus toxoid were used as positive controls. Production of interferon (IFN)-γ and interleukin (IL)-4 upon antigen stimulation in vitro was measured using the sandwich ELISA technique. Blood was processed within 4 hours of sampling and the samples were shipped at a temperature of 4°C (on ice) in 50-mL Falcon tubes. The sample size of approximately 8 per group was selected to obtain preliminary data on the effect of the vaccine on antibody and cellular immune responses at different dose levels and schedules in H pylori-negative subjects. The results of all assays were summarized by collection time points. Geometric mean titers (GMT) at each bleeding and the proportion of subjects with concentrations above particular cut-off values were calculated. The log-transformed data were analyzed by the 1-way analysis of variance method (pairwise comparisons obtained from t tests). The HP3 vaccine was well-tolerated. As expected for an alum-adjuvanted vaccine, the most common local reactions that occurred within 6 days postinjection for both vaccine and placebo were injection site pain and erythema (Table 2). Erythema and induration >25 mm and severe pain occurred at a higher rate on the weekly schedule compared with the monthly schedule. For the monthly schedules, only 1 subject (3%) experienced severe pain and no subject experienced erythema or induration >25 mm. The most common systemic reactions within 6 days postinjection were fatigue, headache, and malaise. Severe reactions were infrequent (only 1 subject reported fever >38.6°C) and were similar in the control and vaccine arms. The safety profile remained unchanged after the booster dose 18–24 months later. Vaccination did not alter laboratory parameters.Table 2Comparison of Frequency (n/%) of Local and Systemic Reactions Within 6 Days Postinjection Occurring During Primary SeriesTreatment groupReaction1 M012L⁎Monthly schedule 0, 1, 2; low dose (10 μg). (n = 7), n (%)2 M012H‡Monthly schedule 0, 1, 2; high dose (25 μg). (n = 7), n (%)3 M014L§Monthly schedule 0, 1, 4; low dose (10 μg). (n = 7), n (%)4 M014H∥Monthly schedule 0, 1, 2; high dose (25 μg). (n = 8), n (%)5 Plac¶Monthly schedule; placebo. (n = 9), n (%)6 W012L#Weekly schedule 0, 1, 2; low dose (10 μg). (n = 9), n (%)7 W012H⁎⁎Weekly schedule 0, 1, 2; high dose (25 μg). (n = 10), n (%)Local Erythema Any2 (29)1 (14)3 (43)2 (25)2 (22)6 (67)7 (70) >25 mm001 (14)003 (33)2 (20) Induration Any4 (56)01 (14)1 (13)06 (67)6 (60) >25 mm000004 (44)3 (30) Pain Any7 (100)7 (100)5 (71)7 (88)6 (67)9 (100)9 (90) Severe1 (14)00002 (22)5 (50)Systemic Malaise Any2 (29)4 (57)2 (29)3 (38)6 (67)8 (89)4 (40) Severe1 (14)1 (14)1 (14)1 (13)1 (11)00 Myalgia Any1 (14)1 (14)2 (29)2 (25)3 (33)5 (56)2 (20) Severe01 (14)00000 Arthralgia Any1 (14)2 (29)2 (29)001 (11)0 Severe0000000 Nausea Any2 (29)3 (43)1 (14)01 (11)2 (22)1 (10) Severe001 (14)0000 Headache Any2 (29)5 (71)3 (43)1 (13)5 (56)7 (78)6 (60) Severe2 (29)2 (29)1 (14)001 (11)2 (20) Chills Any01 (14)1 (14)1 (13)02 (22)0 Severe001 (14)0000 Fatigue Any4 (57)5 (71)4 (57)2 (25)5 (56)9 (100)5 (50) Severe1 (14)2 (29)1 (14)1 (13)003 (30) Rash Any1 (14)01 (14)01 (11)01 (10) Urticarial0000000 Fever Any00001 (11)1 (11)1 (10) >37.8°–38.5°C1 (11)01 (10) 38.6°–39.5°C01 (11)0 Stayed home1 (14)01 (14)1 (13)02 (22)1 (10) Analgesic/use of antipyretics2 (29)2 (29)2 (29)1 (13)2 (22)3 (33)4 (40) Monthly schedule 0, 1, 2; low dose (10 μg).‡ Monthly schedule 0, 1, 2; high dose (25 μg).§ Monthly schedule 0, 1, 4; low dose (10 μg).∥ Monthly schedule 0, 1, 2; high dose (25 μg).¶ Monthly schedule; placebo.# Weekly schedule 0, 1, 2; low dose (10 μg). Weekly schedule 0, 1, 2; high dose (25 μg). Open table in a new tab Vaccination with HP3 at both low (10 μg) and high (25 μg) doses induced a strong antibody response to all 3 antigens that peaked 1 month after the third dose. Antibody responses in subjects receiving the vaccine on months 0, 1, and 2 were low after the first immunization, and increased substantially 1 month after the second and the third immunizations (Figure 1). Antibody titers decreased during the 3-month follow-up period, but remained above the cut-off levels. Antibody responses were similar after the first and the second doses in subjects vaccinated at months 0, 1, and 4. However, GMTs decreased after the second injection, in some cases to cut-off levels (Figure 1). The third injection induced a substantial increase of the GMT to each of the 3 antigens, suggesting a boosting effect of the third dose of the vaccine on the antibody response primed by the first 2 doses. For both dosing schedules antigen-specific antibody responses in vaccinated groups were significantly higher than placebo (P = .0001) 1 month after the third dose. There were no statistically significant differences in GMTs between low and high dosages for the 2 dosing schedules. Subjects vaccinated weekly with both low and high doses developed a strong antibody response to VacA after the second dose, whereas weaker responses were observed for NAP and CagA (Supplemental Figure 1; available online at www.gastrojournal.org). Antigen-specific antibody responses peaked 1 month after the third vaccination. At follow-up month 5 (4.5 months after the third immunization), antibody titers to each of the antigens were decreased but still above the cut-off levels for NAP and VacA and borderline for CagA. The levels of antigen-specific antibodies in the serum were paralleled by the proportion of individuals who seroconverted to ≥1 of the antigens (Figure 2). One month after the third dose, irrespective of immunization schedule or dosage, all individuals had seroconverted to at least 2 of the 3 antigens (in most cases VacA and NAP) and 86%–90% of subjects se