Epstein–Barr Virus Vaccines

Subunit gp350 Vaccines

Two subunit prophylactic gp350 EBV vaccines have been evaluated in human subjects. The first vaccine, developed by Gu and colleagues, was created by transfecting human embryonic lung cells with a vaccinia virus construct from the smallpox vaccine strain Tien Tan , which contained the major EBV membrane antigen (EBV-MA). The majority of this material, we now know, was gp350. Cells demonstrating cytopathic effect were harvested and the supernatant from these lysed cells was found to contain high titers of infectious recombinant virus.

After demonstrating that the candidate vaccine was immunogenic in rabbits, the investigators conducted a series of three clinical trials. The first study assessed safety by inoculating 11 healthy adults who had previously received the Tien Tan vaccine. Three vaccine recipients had febrile reactions and local vaccinia lesions with antibody responses to vaccinia. The other eight subjects had mild inflammatory reactions at the scarification sites but no antibody responses to vaccinia. None of the 11 volunteers had a rise in antibody titers to EBV. In the second trial, six school children ages 8 and 9 years who were latently infected with EBV and had previously received Tien Tan vaccine were administered the EBV vaccine. The vaccine was safe, and 1 month after vaccination, eight of the nine developed antibodies to EBV-MA and to vaccinia virus. None had a rise in antibodies against EBV viral capsid antigen (VCA), which meant that the response to EBV-MA was not due to reinfection by EBV circulating in the community.

The final trial enrolled 19 EBV-naïve children who were 1.7–2.8 years old and who had not received Tien Tan vaccine previously. Nine children were vaccinated while 10 served as unvaccinated controls. The vaccine was safe, and all nine vaccinees developed antibodies to EBV-MA and vaccinia. During 16 months of follow-up, 3 of 9 vaccinees and 10 of 10 in the control group became infected with EBV as evidenced by development of antibodies against EBV VCA ( P = 0.003, Fisher exact test, two-sided). This supports the authors’ conclusion that “it has been shown for the first time that protection against and/or delay of EBV infection by the natural route is possible in humans.” The authors state that “live vaccinia vectors can be used and are efficacious.” However, a live vaccinia vector in a prophylactic vaccine is unlikely to be acceptable today given the severe adverse events associated with using smallpox vaccine to enhance bioterrorism preparedness.

The second subunit prophylactic gp350 EBV vaccine that has been tested in human subjects was developed by Jackman and colleagues. They reported the successful production of a recombinant gp350 in Chinese hamster ovary cells and showed by antibody and CD21 binding studies that it closely resembled native gp350. This construct administered with Freund’s adjuvant or alum elicited gp350 and neutralizing antibodies in rabbits. An EBV vaccine produced by this technology has been subsequently evaluated in four clinical trials.

Two sequential double-blind, randomized controlled trials were reported by Moutschen et al. The first was a Phase 1 study evaluating safety and immunogenicity of gp350 vaccine in 67 volunteers (mean age, 21.4 years; range, 18–25 years). The subjects were stratified by EBV antibody status and then randomized to receive 50 μg of gp350/0.5 mL adjuvanted with either 3-O-desacyl-4′-monophosphoryl lipid A and aluminum salt (AS04) or aluminum salt alone. Three doses of vaccine were administered intramuscularly at 0, 1, and 6 months.

One severe adverse event was noted 10 days after receiving a second dose of gp350 adjuvanted with AS04. This previously EBV antibody-positive subject developed severe flu-like symptoms with headache and meningismus followed by arthritis of the knees, ankles, and lower back. The subject fully recovered within 2 months. The authors wrote that “a causal relationship with the vaccine could not be excluded.”

Fifty-one of the subjects participating in the Phase 1 study were analyzed for immunogenicity. All 25 antibody-negative subjects had a gp350 antibody response when tested 1 month after the third dose of vaccine. The geometric mean titer (GMT) was significantly higher among the subjects given ASO4-adjuvanted vaccine as compared with those who received alum as the adjuvant (727 vs 498). All 26 antibody-positive subjects experienced at least a fourfold rise in gp350 antibody titers, with the GMT slightly higher among those subjects given AS04-adjuvanted vaccine (1188 vs 1076). Neutralizing antibody responses were seen in 100% of antibody-negative subjects who received AS04-adjuvanted vaccine as compared with 44% among the antibody-negative subjects given gp350 with alum. Among the antibody-positive subjects, fourfold boosts in neutralizing antibody titers occurred in 71% of those given AS04-adjuvanted vaccine as compared with 56% given alum as the adjuvant. Thus, in the Phase 1 trial, gp350 adjuvanted with AS04 was consistently more immunogenic than gp350 adjuvanted with alum.

In the Phase 1/2 study, 81 EBV-naïve subjects (mean age, 21.8 years; range, 18–37 years) were randomized 1:1:1 to receive unadjuvanted vaccine, vaccine adjuvanted with AS04, or vaccine adjuvanted with alum. Like the Phase 1 study, vaccine was administered as a 50 μg of gp350/0.5 mL dose intramuscularly at 0, 1, and 6 months.

The vaccine was considered to be safe, but one notable reaction was reported. A subject with a history of migraine experienced a severe headache associated with vomiting 27 days after receiving the second dose of vaccine. Because of the vaccinee’s past history of migraine, the relationship of migraine with the vaccination was classified as unlikely.

All 71 evaluable subjects had gp350 antibodies when tested 1 month after the third vaccination. The GMTs were as follows: unadjuvanted vaccine, 258; alum-adjuvanted, 1053; AS04 adjuvanted, 1251. The GMT of the group that received unadjuvanted vaccine was statistically significantly lower than the GMTs of the other two groups. A similar trend was noted for neutralizing antibodies assayed either by inhibition of EBV-infected B cell proliferation or by a competition immunoassay that measured the ability of vaccine-induced antibody to block binding of a murine anti-gp350 monoclonal antibody (72A1) to gp350.

In summary, the Phase 1/2 trial of the gp350 vaccine confirmed the immunogenicity results of the Phase 1 study showing that AS04 was a better adjuvant than alum. The Phase 1/2 study also indicated that unadjuvanted gp350 was inferior to the adjuvanted preparations.

The third trial was a Phase 2, placebo-controlled, double-blind study evaluating safety, immunogenicity, and efficacy of recombinant gp350 vaccine in EBV-naïve young adults ages 16–25 years. The vaccine contained 50 μg of gp350 and 50 μg of AS04 in a 0.5 mL volume. Three doses of vaccine were given intramuscularly at 0, 1, and 5 months. The primary endpoint was the incidence of infectious mononucleosis disease in vaccine and placebo groups 18 months after administration of the second dose of clinical trial material as documented by clinical criteria along with EBV antibody responses to nonvaccine antigens. EBV infection was defined as development of EBV antibody responses to nonvaccine antigens in the absence of clinical infectious mononucleosis. The study enrolled 181 subjects (mean age, 20.6 years; 52% male; 97% white) who were assigned to vaccine or placebo in a 1:1 ratio. No serious adverse events were reported. Almost all (76/77; 98.7%) of the vaccine recipients who were not subsequently infected by wild-type EBV developed gp350 antibodies. The 72A1 competition immunoassay was employed to detect neutralizing antibody. Neutralizing antibody levels peaked 1 month after the third dose “with 69.86% (95% CI, 58.00–80.06%) showing seroconversion at that point in time.” Presumably, this pertained only to the vaccine recipients.

During the 19-month follow-up period, EBV vaccine did not prevent infection: 13 (14%) of 90 vaccine recipients became infected versus 18 (20%) of 91 placebo subjects. However, vaccine had a significant effect on the development of clinical disease. In the intent-to-treat population, infectious mononucleosis developed in only 2 (2%) of 90 vaccinees as compared with 9 (10%) of 91 placebo recipients ( P = 0.03, Fisher exact test, one-sided).

This study is noteworthy in that it was the first to demonstrate protection against clinical infectious mononucleosis by an EBV subunit vaccine. However, it had limitations. EBV viral loads were not measured, the neutralizing antibody results were difficult to interpret, and there was no assessment of EBV-specific cell-mediated immune responses.

The fourth study was a Phase 1 trial of recombinant gp350 vaccine with an aluminum hydroxide adjuvant given to 16 pediatric renal transplant candidates. Subcutaneous dosing regimens of 12.5 μg or 25 μg of gp350 given three or four times (weekly for the first three doses with a fourth dose at week 30–32 if not yet transplanted) over a total of 32 weeks were well tolerated. Three subjects were excluded because they did not complete the two immunogenicity evaluations. All 13 evaluable subjects had an anti-gp350 antibody response, but only 4 subjects made neutralizing antibody. Because there was no control group, vaccine efficacy could not be evaluated. This small Phase 1 trial showed that immunization of children awaiting transplantation for chronic renal disease is feasible, but the low amounts of adjuvanted gp350 given were proba­bly insufficient to elicit optimal immune responses.