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A Canarypox Vector Expressing Cytomegalovirus (CMV) Glycoprotein B Primes for Antibody Responses to a Live Attenuated CMV Vaccine (Towne)

  1. Stuart P. Adler1,
  2. Stanley A. Plotkin2,
  3. Eva Gonczol3,4,
  4. Michel Cadoz5,
  5. Claude Meric5,
  6. Jian Ben Wang1,
  7. Pierre Dellamonica6,
  8. Al M. Best1,
  9. John Zahradnik2,
  10. Steve Pincus7,
  11. Klara Berencsi3,
  12. William I. Cox7 and
  13. Zsofia Gyulai3
  1. 1Medical College of Virginia/Virginia Commonwealth University, Richmond
  2. 2Pasteur Mérieux Connaught USA, Swiftwater
  3. 3The Wistar Institute, Philadelphia, Pennsylvania
  4. 4Albert Szent-Gyorgyi Unversity, Szeged, Hungary
  5. 5Pasteur Mérieux Connaught France, Marcy l'Étoile
  6. 6Hôpital de l'Archet, Nice, France
  7. 7Virogenetics, Troy, New York
  1. Reprints and correspondence: Dr. Stuart P. Adler, Medical College of Virginia, P.O. Box 980163, Richmond, VA 23298-0163 (SADLER{at}HSCVCU.EDU).
 
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Abstract

To develop a vaccine against cytomegalovirus (CMV), a canarypox virus (ALVAC) expressing CMV glycoprotein (gB) was evaluated alone or in combination with a live, attenuated CMV vaccine (Towne). Three doses of 106.5 TCID50 of ALVAC-CMV(gB) induced very low neutralizing or ELISA antibodies in most seronegative adults. However, to determine whether ALVAC-CMV(gB) could prime for antibody responses, 20 seronegative adults randomly received either 106.8 TCID50 of ALVAC-CMV(gB) or 106.8 TCID50 of ALVAC-RG, expressing the rabies glycoprotein, administered at 0 and 1 month, with all subjects receiving a dose of 103.5 pfu of the Towne vaccine at 90 days. For subjects primed with ALVAC-CMV(gB), neutralizing titers and ELISA antibodies to CMV(gB) developed sooner, were much higher, and persisted longer than for subjects primed with ALVAC-RG. All vaccines were well tolerated. These results demonstrate that ALVAC-CMV(gB) primes the immune system and suggest a combined-vaccine strategy to induce potentially protective levels of neutralizing antibodies.

A live, attenuated cytomegalovirus (CMV) vaccine (Towne) has been evaluated in normal volunteers and in renal transplant patients [1, 2]. Towne was immunogenic, reduced the severity and incidence of CMV-associated disease among seronegative recipients of seropositive kidneys [2], and did not reactivate in these patients [3]. Although Towne protected against low doses of an unattenuated strain of CMV in an artificial challenge study, at a reduced dose that induced low neutralizing titers, Towne failed to prevent child-to-mother transmission of CMV, whereas naturally seropositive women were protected [4, 5].

Avipox viruses, such as canarypox (ALVAC), are candidate vaccine vectors because the ALVAC genome accepts large amounts of foreign DNA and will direct the synthesis of multiple foreign proteins. ALVAC does not produce progeny in mammalian cells or nonavian species. It does, however, elicit protective immune responses in nonavian species [6, 7]. Moreover, live ALVAC does not produce disease in healthy subjects or immunosuppressed patients [8].

To develop a vaccine strategy for obtaining high levels of neutralizing antibodies against CMV in humans, we evaluated the safety and immunogenicity of an ALVAC recombinant expressing the major CMV envelope glycoprotein (gB) given alone or in combination with Towne vaccine.

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Materials and Methods

Trial A

The first study, conducted at the Hôpital de l'Archet (Nice, France), enrolled 20 healthy male and female volunteers (10 seronegative and 10 seropositive), 18–50 years old. By use of an open-label format, each volunteer received three doses of ALVAC-CMV(gB) vaccine (batch S2723), each dose containing 106.5 TCID50 of canarypox. The second and third doses were administered 28 and 180 days after the initial dose. Volunteers were monitored for 30 min after each injection and then were examined by a study physician on days 1, 2, 3, and 7 after each injection, to monitor local and systemic reactions. Blood samples were taken on days —14, 0, 2, 7, 28, 30, 35, 56, 180, 182, 187, and 208. Samples of saliva and urine were taken on days —14, 0, 7, 28, 35, 56, 180, 187, and 208.

Trial B

The second vaccine study, conducted in Richmond, VA, enrolled 20 healthy CMV-seronegative male and female volunteers, 20–43 years old. By use of a double-blind format, volunteers were randomized to receive either an initial injection of 106.8 TCID50 ALVAC-CMV(gB) (batch S3145) and a second, similar injection 1 month later or a first injection of 106.8 TCID50 of ALVAC-RG (batch S3106) and a second, similar injection 1 month later. Both groups received a single dose of 103.5 pfu of Towne vaccine 90 days after the initial dose of ALVAC vaccine. Samples of blood, urine, and saliva were collected for each subject on days 30, 60, 90, 97, 114, 120, 180, 270, and 360 after immunization. Two subjects, both assigned to the ALVAC-RG group, withdrew from the study prior to completion for reasons unrelated to study participation. The data on their immune responses were not included.

All Richmond subjects were monitored for vaccine-associated illness: (1) the subjects maintained a temperature chart for 2 weeks after vaccination; (2) the subjects were contacted by phone by a research nurse 3 days after each vaccine administration; (3) the subjects self-reported (via a diary card) any acute local or systemic reaction, including any pain, swelling, or erythema; and (4) at follow-up visits, an interval history was obtained.

Vaccines

The Towne vaccine (lot C-107) was prepared by Program Resources (Rockville, MD). The seed strain was Towne pool, passage 131. The vaccine pool was prepared in cultures of MRC-5 cells. A harvest pool was clarified by centrifugation at 1200 g for 20 min and then aliquoted into 3 mL vials for lyophilization. The vials were stored at 2°C–8°C for 19 days, then at −20°C, andfinally at −70°C.

The mean infectivity titer was 103.5 pfu/lyophilized vial (0.5 mL), which constituted a single dose. Before use, the vaccine was reconstituted in sterile water, maintained on ice, and administered subcutaneously in the deltoid region within 1 h of reconstitution.

ALVAC was developed by Virogenetics (Troy, NY) and manufactured by Pasteur Mérieux Connaught (Marcy l'Étoile, France). ALVAC-CMV(gB), laboratory designation vCP139, is a recombinant canarypox virus encoding the full-length gB gene of the Towne strain of CMV, whereas ALVAC-RG, laboratory designation vCP65, expresses the glycoprotein G gene of the ERA strain rabies virus. Both vaccines were prepared in primary chick embryo fibroblasts derived from specific pathogen-free eggs. Clarified lysates of infected cells were diluted in serum-free virus stabilizer and lyophilized. Titrations were performed on QT35 cells by the endpoint dilution method. Batches S3106 (ALVAC-RG), S2723 (ALVAC-CMV[gB]), and S3145 (ALVAC-CMV[gB]), with titers of 106.8,106.5, and 106.8 TCID50 per dose, respectively, were used in the trials. Both recombinants were tested in small animals [9], and ALVAC-RG was previously tested in humans and has an extended record of safety [8]. The lyophilized preparations were reconstituted in 1.0 mL of sterile water. The vaccines were given by injection (1.0 mL) into the deltoid muscle within 15 min of reconstitution.

Laboratory methods

Seronegativity prior to enrollment was established by measuring IgG to CMV by use of an enzyme immunoassay, as described elsewhere [10]. Neutralizing antibodies were determined by a standard reduction assay [11]. A titer was defined as the reciprocal of the highest dilution of serum that fully inhibited viral cytopathic effect (CPE), compared with 100% CPE in control wells.

IgG antibodies to gB in sera were measured by an enzyme immunoassay, as described elsewhere [12]. Antibodies to the canarypox vector (ALVAC) were measured at Pasteur Mérieux Connaught by use of an enzyme immunoassay with plates coated with purified ALVAC-CPpp virus (ALVAC vector without insert).

CMV-specific lymphocyte responses were determined as described elsewhere [13]. Lymphocyte proliferation responses were expressed as stimulation indices, defined as the ratio of counts per minute (CPM) in CMV antigen-stimulated cultures to CPM in control antigen-stimulated cultures. To measure lymphocyte responses to CMV(gB), purified gB was used at 3.0 µg/mL.

Urine and saliva samples of trial B were cultured, before and after concentration, in duplicate on MRC-5 fibroblasts, and white blood cells were prepared from each plasma sample and cultured for CMV.

Statistical analysis

Groups were compared with repeated-measures analysis of variance assessing group versus time interactions. For antibody data, the log of the reciprocal titers was analyzed. A neutralizing titer of 1 was attributed to sera showing incomplete neutralization at the lowest dilution tested (1 : 4 or 1 : 8).

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Results

Safety and reactogenicity

No severe adverse reactions occurred in either trial. Local and systemic reactions were mild, consisting mainly of pain and redness at the injection site. All three immunizations were well tolerated in all recipients. Seropositive and seronegative subjects had the same frequency of local and systemic reactions.

In trial B, for the first two doses, reactogenicity was mild, and both ALVAC-CMV(gB) and ALVAC-RG were well tolerated. Towne vaccine (dose 3) induced a frequency of local or systemic reactions similar to that for the ALVAC vaccines. No subject had Towne vaccine virus recovered in urine, saliva, or blood.

Immunogenicity

In trial A, three doses of ALVAC-CMV(gB) administered on days 0, 28, and 180 failed to increase neutralizing titers among the 10 seropositive subjects or to induce significant neutralizing titers among the 10 seronegative subjects. No intercurrent CMV infections occurred.

We performed a second trial to determine whether ALVAC-CMV(gB), while not eliciting detectable levels of antibodies itself, could prime an immune response to gB. Two groups were randomly assigned to receive two injections, 1 month apart, of either ALVAC-CMV(gB) (10 volunteers) or ALVAC-RG (8 volunteers). Both groups received a single injection of Towne vaccine 90 days after the first ALVAC injection.

After two injections of ALVAC and before booster with Towne on day 90, a low increase (P < .057) in anti-gB ELISA titers was observed in ALVAC-CMV(gB) recipients, whereas no change was observed in the neutralizing titers. No change in either anti-gB ELISA or CMV-specific neutralizing antibodies was observed in ALVAC-RG recipients (table 1).

Table 1
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Table 1

ELISA antibody responses to cytomegalovirus gB and neutralizing titers in trial B.

After administration of the Towne vaccine on day 90, the ALVAC-CMV(gB) group developed significantly higher mean ELISA titers against CMV gB [F(9144) = 4.03, P < .0001] and mean neutralizing titers [F(9142) = 8.9, P < .0001] than the ALVAC-RG group. The kinetics of the responses of the two groups were also different: in the ALVAC-CMV(gB) group, the ELISA anti-gB response increased as early as day 7 and the neutralizing response as early as day 14 after Towne booster, whereas 28 days were needed to obtain a response, measured by either ELISA or neutralizing titers, in the ALVAC-RG recipients. The titers also persisted at significantly higher levels up to day 360 in the ALVAC-CMV(gB) group, compared with the ALVAC-RG group (table 2). An IgG antibody response to the canarypox vector was induced in all vaccinees in both trials (data not shown).

Table 2
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Table 2

Lymphoproliferative responses to either cytomegalovirus gB antigen or Towne antigen in trial B.

Priming with ALVAC-CMV(gB) allowed the Towne vaccine to induce peak ELISA titers to gB and CMV neutralizing titers at levels significantly higher than those observed among the naturally seropositive subjects in trial A. For the naturally seropositive subjects at entry (day 0), the geometric mean ELISA antibody titer to gB was 1 : 25591, compared with a peak (180 days) geometric mean titer of 1 : 89321 for the subjects primed with ALVAC-CMV(gB) in trial B (P = .03). The same was true for neutralizing titers. At entry, the naturally seropositive subjects in trial A had a geometric mean neutralizing titer of 1 : 79, compared with a peak (180 days) geometric mean titer of 1 : 259 for the subjects primed with ALVAC-CMV(gB) in trial B (P = .008).

Lymphoproliferative responses

Lymphoproliferative responses were measured monthly in trial B over the first 180 days in each subject by use of two antigens: purified gB and extracts of Towne CMV—infected cells. There were no statistically significant differences in the mean lymphoproliferative responses between the group primed with ALVAC-CMV(gB) and the group primed with ALVAC-RG (table 2).

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Discussion

The results of these trials demonstrated that an ALVAC recombinant containing the gene coding for CMV gB could prime the immune system to produce a strong neutralizing response to the gB produced by an attenuated CMV virus.

Like other ALVAC recombinants tested in humans, the ALVAC-CMV(gB) vaccine was well tolerated: only mild local reactions and minimal systemic reactions were observed. These reactions were similar to those observed with the CMV Towne vaccine.

The very low levels of gB-specific IgG and neutralizing activities induced by ALVAC-CMV(gB) in humans were unexpected, because this vaccine induces detectable levels of these activities in mice and guinea pigs [9]. Mammalian cells support only an abortive infection by canarypox, with viral gene expression limited to only early genes and for a relatively short duration. As there is no evidence that expression of CMV gB is lower in humans than in small mammals, CMV gB may be a poorer immunogen in humans than in laboratory animals. The observation that two immunizations of ALVAC-CMV(gB) induced a low immune response in trial B but none in trial A can probably be explained by a difference in the titer of the two batches of vaccine used.

An important observation made in this trial is that this ALVAC vaccine could prime for an immune response to a live, attenuated vaccine (Towne) and induced peak ELISA anti-gB titers and neutralizing titers at levels equal to or higher than those observed among the naturally seropositive subjects.

For CMV, the majority of the immunogenic neutralizing epitopes occur on the gB envelope glycoprotein of the viral particle [14], and the data of the current study are consistent with this, as both antibodies to CMV gB and neutralizing activity were boosted to high levels after priming with CMV gB.

Lymphoproliferative responses to the Towne vaccine were not primed by gB. We previously found that induction of maximal lymphoproliferative responses to Towne antigen required less antigen than that required to induce maximal antibodies, suggesting that the Towne vaccine is an effective inducer of lymphoproliferative responses [5]. The lack of priming of lymphoproliferative responses by ALVAC-CMV(gB) suggests that gB expressed in ALVAC-infected cells is an inefficient inducer of lymphoproliferative responses.

Results of this trial confirm similar trials using ALVAC expressing HIV-1 MN gp160 [15]: ALVAC alone induces a weak antibody response but primes for subsequent exposure to HIV envelope.

We previously evaluated the ability of the Towne strain of CMV to prevent the child-to-mother transmission of CMV [5]. Women who were naturally seropositive appeared to be protected from acquiring a CMV infection, but women who received the Towne vaccine were unprotected. In this previous trial, the Towne vaccine was used at a very low dose and produced neutralizing titers that were 10- to 20-fold lower than those produced by a wild-type infection. Furthermore, we observed that naturally seropositive adults and those with vaccineinduced immunity who had serum neutralizing titers ⩾1 : 64 also had detectable levels of IgG antibodies to CMV gB in nasal washes and saliva [12]. Because gB contains the majority of neutralizing epitopes, we proposed that serum neutralizing titers >1 : 64 would be necessary for protection against wild-type infection [12]. The current trial has demonstrated the feasibility of using low doses of ALVAC-CMV(gB) to prime the immune system to the gB protein and obtain enhanced antibody responses and neutralizing titers >1 : 64. These results define a vaccine strategy to induce potentially protective levels of neutralizing antibodies. The recall antigen could be either a live virus or, potentially, a subunit antigen.

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Acknowledgments

We thank the study participants; Raphaëlle El Habib, Maurice Harmon, and Ken Guito for the preparation of the regulatory documents; Maurice Raux and Christine Blondeau for ALVAC serologies; Rae Lyn Burke for supplying recombinant gB protein; Olivier Level and Monique Ollivier for the preparation of the ALVAC clinical batches; Linda Petro and Géraldine Lancelot for monitoring the trials; Bernard Meignier and Jim Tartaglia for continuous support; and Susan Wood for revising the manuscript.

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Footnotes

  • Informed consent was obtained from all subjects. The human experimentation guidelines of the US Department of Health and Human Services and those of Medical College of Virginia/Virginia Commonwealth University were followed in the conduct of the clinical research. The protocol was approved by the Comité Consultatif pour la Protection des Personnes dans la Recherche Biomédical (CCPPRB) of Nice, France.

  • This work was supported by Pasteur Mérieux Connaught.

  • Received January 6, 1999.
  • Revision received April 22, 1999.
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  1. J Infect Dis. (1999) 180 (3): 843-846. doi: 10.1086/314951
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