Table 4.

Characteristics of the clinical studies included in the systematic review.

First author, year of publication, and title (glycoprotein[s] targeted)	Location, Phase and Vaccine	Study endpoints and Study population features	Study Groups and Features	Measurement of vaccine response	Trial Outcomes	Overall assessment
1. Rees, 2009 (80) A Phase I trial of Epstein-Barr virus gp350 vaccine for children with chronic kidney disease awaiting transplantation (gp350)	United Kingdom, Phase I, open fixed-dose escalation Recombinant gp350 protein (splice-site mutated to prevent formation of 220 isoform) produced in CHO cells	Safety and immunogenicity 16 EBV-negative children with chronic kidney disease (CKD) awaiting renal donation (median age 8.9 years, range 1.4-17.6 years)	Groups: Immunization with 12.5µg gp350 adjuvanted with alhydrogel (n=6); 25µg gp350 adjuvanted with alhydrogel (n=10). Immunization Schedule: Three three- or four-weekly subcutaneous injections. A fourth vaccination was offered at weeks 30 to 32 for EBV-negative children who were not transplanted and had low anti-gp350 antibody levels. Sample collection Schedule: Variable (not defined) Study Duration: ~40 weeks	Antibody assays: ELISA (gp350). In vitro neutralization: Competitive ELISA (gp350) against anti-gp350 neutralizing antibody (not reported) using sera from immunized individuals. Measurement of in vivo infection: ELISA (EBNA1, VCA), immunoblot (antigens not reported), PCR (EBNA1, BamHI-W).	The vaccine was well-tolerated at both dose levels, with only two systemic reactions occurring at the 25µg dose level. Two patients received only two vaccinations (group(s) unclear). In the 25µg dose level group, 3/10 patients received a fourth vaccination at week 30-32. All evaluable patients in the 12.5µg dose level (n=4) and 25µg dose level (n=9) displayed a detectable anti-gp350 response. Only 1/4 patients in the 12.5µg dose level 3/9 patients in the 25µg dose level displayed nAb responses. The n=1 evaluable patient that received a fourth vaccination experienced a rapid and substantial increase in anti-gp350 antibodies, and the emergence of nAb, which were absent before. Four patients (group(s) unclear) acquired EBV infection during the study period, and seven others were reported to display high levels of EBV genomic loads 26 weeks post-transplant (off-trial), with levels similar to unvaccinated transplant recipients that had been EBV-negative prior to transplant, which were higher that unvaccinated transplant recipients that had been EBV-positive prior to transplant. One episode of EBV-related PTLD was reported in a patient in the 12.5µg dose level soon after transplantation, 50 weeks after the first vaccination.	Results from this study confirm this gp350-based vaccine at the current formulation and doses is safe and tolerable in children with CKD awaiting transplantation. The vaccine was immunogenic in all evaluable patients, but only 4/13 generated nAbs. EBV infection was detected in various patients both before and after transplantation, with one case of EBV-associated PTLD. Although the study was not designed to test vaccine efficacy, these results suggest the vaccine does not influence post-transplant EBV loads or prevent PLTD at its current state. Thus, new formulations and/or vaccination schedules might be needed to successfully lower EBV loads post-transplant the occurrence and severity of EBV-associated PTLD in this patient population.
2. Sokal, 2007 (81) Recombinant gp350 vaccine for infectious mononucleosis: a Phase 2, randomized, double- blind, placebo-controlled trial to evaluate the safety, immunogenicity, and efficacy of an Epstein- Barr virus vaccine in healthy young adults (gp350)	Belgium, Phase II, double-blinded placebo-controlled randomized Recombinant gp350 protein (splice-site mutated to prevent formation of 220 isoform) produced in CHO cells	Efficacy at preventing infectious mononucleosis (primary); efficacy at preventing primary EBV infection, and immunogenicity (secondary) 181 EBV-negative 16–25-year-old individuals (51.1% male and 97.8% Caucasian, 20.6 years mean age, in vaccine group; 53.8% male and 96.7% Caucasian, 20.5 years mean age, in placebo group)	Groups: Immunization with 50µg gp350 adjuvanted with ASO4 (n=90); 0.5mg Alum (placebo, n=91). Immunization Schedule: Three intramuscular injections at month (0, 1, 5) Sample collection Schedule: Month (0, 1, 5, 6, 19) Study Duration: 18-21 months	Antibody assays: ELISA (gp350). In vitro neutralization: Competitive ELISA (gp350) against anti-gp350 neutralizing antibody 72A1 using sera from immunized individuals. Measurement of in vivo infection: IFA (VCA IgG and IgM) and ELISA (VCA IgG and IgM); health monitoring for infectious mononucleosis symptoms.	The vaccine was well-tolerated, and no severe adverse events associated with the vaccine were reported. In the placebo group, 18/90 of participants became infected, nine who developed infectious mononucleosis and nine who displayed asymptomatic infection. In the vaccine group, 13/90 participates became infected, two who developed infectious mononucleosis (both before the third immunization) and eleven who displayed asymptomatic infection. Infectious mononucleosis rates were found to be significantly different between the two groups, and vaccine efficacy at preventing infectious mononucleosis was reported to be 78% (intention to treat population). 1 month after the third dose, anti-gp350 antibodies were detected in 98.7% of participants who hadn’t already become EBV-positive; it was only at this point that anti-gp350 antibody levels in vaccinees exceeded those seen after natural infection. Neutralizing antibodies as measured by 72A1 competitive ELISA peaked at this time as well, detected in 69.86% of participants.	Results from this study suggest a gp350-based vaccine can be effective at preventing infectious mononucleosis in healthy young adults (78% efficacy). While the study was not designed to measure long-term efficacy, the results suggest the vaccine might protect against infectious mononucleosis even after eighteen months post-primary immunization. In this system, three doses might provide full protection against disease; however, the vaccine was not successful at preventing primary EBV infection.
3. Moutschen, 2007 (82) Phase I/II studies to evaluate safety and immunogenicity of a recombinant gp350 Epstein–Barr virus vaccine in healthy adults (gp350)	Belgium, Phase I, double-blind randomized Recombinant gp350 protein (splice-site mutated to prevent formation of 220 isoform) produced in CHO cells	(a) Safety (primary) and immunogenicity (secondary) 36 EBV-negative and 31 EBV-positive 18–24-year-old young adults (59.7% male, 94.0% Caucasian)	Groups: Immunization with 50µg gp350 adjuvanted with ASO4, EBV-negative (EBV-/AS04, n=20); 50µg gp350 adjuvanted with ASO4, EBV-positive (EBV+/AS04, n=15); 50µg gp350 adjuvanted with Alum, EBV-negative (EBV-/Alum, n=15); 50µg gp350 adjuvanted with Alum, EBV-positive (EBV+/Alum, n=16). Immunization Schedule: Three intramuscular injections at month (0, 1, 6) Sample collection Schedule: Month (0, 1, 2, 6, 7) Study Duration: 7 months	Antibody assays: ELISA (gp350). In vitro neutralization: Neutralization assay (transformation inhibition) in B lymphocytes from adult donors, with sera from immunized individuals. Measurement of in vivo infection: IFA and ELISA (assay details not provided).	Both vaccine formulations were found to be safe and well-tolerated, with only one serious adverse event reported in EBV+/AS04 that was deemed potentially related to vaccination. Anti-gp350 antibodies were detected in all vaccinees one month after third immunization. Neutralizing antibodies were detected in 100% of EBV-/AS04, 71.4% in EBV+/AS04-, 44.4% in EBV-/Alum, and 55.5% of EBV+/Alum vaccinated subjects one month after third immunization.	Results from both studies confirm that recombinant soluble gp350 as a non-adjuvanted or adjuvanted (Allum or AS04) vaccine is safe and immunogenic in both EBV-negative and EBV-positive healthy adults. The vaccine stimulated both humoral and cellular immunity (not discussed here). However, despite all participants generating anti-gp350 antibodies after the third immunization, not all vaccinees developed neutralizing antibodies. Four subjects in the Phase I trial, and six subjects in the Phase I/II trial became EBV positive as detected during a 7-month follow-up.
	Belgium, Phase I/II, double-blind randomized Recombinant gp350 protein (splice-site mutated to prevent formation of 220 isoform) produced in CHO cells	(b) Safety (primary) and immunogenicity (secondary) 81 EBV-negative 18–36-year-old adults (55.6% male and 98.8% Caucasian)	Groups: Immunization with 50µg gp350 adjuvanted with Alum (n=27); 50µg gp350 adjuvanted with AS04 (n=27); 50µg non-adjuvanted gp350 (n=27). Immunization Schedule: Three intramuscular injections at month (0, 1, 6) Sample collection Schedule: Month (0, 1, 2, 6, 7) Study Duration: 7 months	Antibody assays: ELISA (gp350). In vitro neutralization: Neutralization assay (transformation inhibition) in B lymphocytes from adult donors, with sera from immunized individuals; competitive ELISA (gp350) against anti-gp350 neutralizing antibody 72A1 using sera from immunized individuals. Measurement of in vivo infection: IFA (VCA IgG and IgM) and ELISA (VCA IgG and IgM).	All vaccine formulations were found to be safe and well-tolerated, with only 1 serious adverse event reported in the Alum-adjuvanted group, but it was not suspected to be related to vaccination. Anti-gp350 antibodies were detected in all vaccinated subjects 1 month after third immunization, but non-adjuvanted vaccine resulted in lower titers. Neutralizing antibodies were detected in 60.9% of Alum-adjuvanted vaccinees, 50% of AS04-aduvanted vaccinees, and 32% of non-adjuvanted vaccinees, as measured by neutralization assay, and in 81.8% of Alum-adjuvanted vaccinees, 71.4% of AS04-aduvanted vaccinees, and 50% of non-adjuvanted vaccinees, as measured by competitive ELISA.
4. Gu, 1995 (83) First EBV vaccine trial in humans using recombinant vaccinia virus expressing the major membrane antigen (gp350)	China, Phase I Live recombinant vaccinia virus (Tien Tan strain) expressing gp220-340 produced in 2BS human cells	(a) Safety and immunogenicity 11 EBV-positive and vaccinia-exposed adults	Groups: Immunization with 108 pfu/ml of vaccinia virus expressing gp220-340 (n=11). Immunization Schedule: Once, by scarification at two sites (single arm) Sample collection Schedule: Month (0, 1) Study Duration: 1 month	Antibody assays: IFA and ELISA (vaccinia, EBV-gp350, and EBV VCA). In vitro neutralization assays: NA Measurement of in vivo infection: IFA and ELISA antibody assays.	3/11 vaccinees had fever reactions and local lesions, with seroconversion to vaccinia. 8/11 had weak responses with local redness, swelling and itching, but no seroconversion to vaccinia. There were no detectable differences in anti-gp350 antibody before and after immunization in all vaccinees.	Results from the adult study suggest that previous exposure to vaccinia virus impaired immune response against the vaccine. Vaccinia non-exposed juveniles and infants displayed an overall strong response to the vaccine. However, based on the infant study, the vaccine might be effective at reducing rate of EBV infection, but not at fully preventing infection.
		(b) Safety and immunogenicity 6 EBV-positive and vaccinia non-exposed 8–9-year-old juveniles	Groups: Immunization with 107 pfu/ml of vaccinia virus expressing gp220-340 (n=6). Immunization Schedule: Once, by a single scarification Sample collection Schedule: At month (0, 1) Study Duration: 1 month	Antibody assays: IFA and ELISA (vaccinia, EBV-gp350, and EBV VCA). In vitro neutralization assays: Neutralization assay using patient sera in Raji cells. Measurement of in vivo infection: IFA and ELISA antibody assays.	Vaccinees developed local lesions with no fever. 5/6 developed antibodies to both vaccinia and gp350 one-month post-immunization. EBV nAbs were detected in sera of all vaccinees between 1:20 and 1:80 dilutions.
		(c) Safety, immunogenicity, and efficacy in preventing primary EBV infection 19 EBV-negative and vaccinia non-exposed 1-3-year-old infants	Groups: Immunization with 107 pfu/ml of expressing gp220-340 (n=9); unvaccinated (n=10). Immunization Schedule: Once, by a single scarification Sample collection Schedule: At month (0, 1, 6, 16) Study Duration: 16 months		Vaccinees developed local lesions with no fever. All vaccinees developed anti-vaccinia antibodies. 8/9 vaccinees developed anti-gp350 antibodies one month after vaccination and nAbs in sera detected between 1:40 and 1:60 dilutions. 3/9 vaccinated infants and 10/10 unvaccinated infants became infected with EBV by Month 16 post-immunization.