Rose Sekulovich

A Subunit Cytomegalovirus Vaccine Based on Recombinant Envelope Glycoprotein B and a New Adjuvant

A phase I randomized, double-blind, placebo-controlled trial was done with a cytomegalovirus (CMV) vaccine based on the envelope glycoprotein, gB, combined with a novel adjuvant, MF59. Participants received CMV gB vaccine with MF59 or CMV gB with alum or placebo at 0, 1, and 6 months. A fourth vaccine was given at 12 months to a subgroup. Levels of neutralizing antibody and antibody to gB 2 weeks after the third dose of vaccine exceeded those in seropositive control subjects. the formulation with MF59 was more immunogenic than that with alum. The optimal dose of gB appeared to be between 5 and 30 microg. The fourth dose produced a prompt rise in antibody level. There were no serious adverse events associated with vaccine. Local and systemic reactions were generally mild and, except for pain at the injection site, occurred with similar frequency in recipients of placebo and CMV vaccine.

Placebo-controlled trial of vaccination with recombinant glycoprotein D of herpes simplex virus type 2 for immunotherapy of genital herpes

Immunotherapy of chronic viral diseases with vaccines is an important but unproven concept. We investigated the effect of a vaccine containing recombinant glycoprotein D (gD2) of herpes simplex virus type 2 (HSV-2) on the frequency of symptomatic outbreaks in patients with genital herpes. 98 patients with documented genital herpes who reported 4-14 recurrences per year were enrolled in a double-blind, placebo-controlled trial. Subjects received injections of either 100 micrograms gD2 in alum or alum alone (placebo) at 0 and 2 months, and recurrences were documented for 1 year. The vaccine was well tolerated. gD2 recipients reported fewer recurrences per month than placebo recipients (mean 0.42 [SE 0.05] vs 0.55 [0.05]; p = 0.055), had fewer virologically confirmed recurrences per month (0.18 [0.03] vs 0.28 [0.03]; p = 0.019), and had a lower median number of recurrences for the study year (4 [range 0-17] vs 6 [0-15]; p = 0.039). Neither genital recurrence nor the placebo vaccine had any discernible effect on HSV-2-specific antibody responses, but gD2 vaccine boosted neutralising antibodies to HSV-2 fourfold and gD2-specific titres sevenfold over baseline levels. These results inspire optimism about the potential use of vaccine for the treatment of chronic, recurring viral diseases.

A Recombinant Glycoprotein Vaccine for Herpes Simplex Type 2: Safety and Efficacy

Genital infections caused by herpes simplex viruses (HSV) are prevalent worldwide [1]. The virus is the major cause of genital ulcerations in Europe and North America [2-4], and seroprevalence studies have shown that between 10% and 20% of European and North American persons aged 20 to 45 years have evidence of HSV-2 infection [1, 5, 6]. Annual acquisition rates of HSV-2 in sexually active populations are estimated to be 1% to 4% [1, 7-10]; among attendees of sexually transmitted disease clinics, the prevalence of HSV-2 seropositivity ranges from 30% to 80% [1, 7]. The incidence of neonatal herpes, one of the major complications of genital herpes, has increased two- to fivefold in the United States and Scandinavia in the last two decades [6, 11]. Vaccines for preventing or treating genital herpes would be a major public health advance. Several observations support the protective effect of preexisting HSV-specific immune responses in limiting the acquisition of HSV-2, the predominant virus type isolated from the genital tract: 1) Reinfection of persons with a second strain of the same viral subtype in the same anatomical area rarely occurs [12-15]; 2) acquisition of neonatal HSV-2 is uncommon among infants exposed to the virus at the time of delivery if the mother is HSV-2-seropositive [16, 17]; and 3) previous HSV-1 infection of the oral-labial region reduces the acquisition and severity of subsequent HSV-2 infection by 40% to 60% [7, 8, 18-22]. Neutralizing antibodies to HSV are predominantly directed to the viral surface glycoproteins, especially to the two essential and abundant glycoproteins D (gD) and B (gB) [23-25]. Monoclonal antibodies to gB and gD protect mice from experimental challenge with HSV [26, 27]. Herpes simplex virus type 2 glycoprotein D (gD2) and B (gB2) have been produced by the use of recombinant DNA technology in Chinese hamster ovary cells as carboxyl terminal-truncated derivatives [28, 29]. These recombinant proteins have been shown to protect guinea pigs from experimental genital challenge with HSV [30-33]. The extent of protection primarily depends on maintaining conformational epitopes of the protein and the potency of the adjuvant used [33, 34]. Immunization with formulations of the two recombinant proteins in the MF59 adjuvant emulsion, which contains squalene, polysorbate 80 (Tween 80, ICI America, Wilmington, Delaware), and sorbitan trioleate (Span 85, ICI America), increases humoral and cellular immune responses and induces protection from experimental challenge in animals [33-35]. Immunization with vaccines containing the recombinant proteins gB and gD combined with the MF59 adjuvant also reduces the rate of subsequent recurrences of genital herpes among previously infected guinea pigs [36, 37]. Our investigations were the initial phase I and II clinical trials to evaluate an HSV-2 subunit vaccine that contained derivatives of gB2 and gD2 in the MF59 adjuvant emulsion and was administered to persons seronegative for HSV or seropositive only for HSV-1 antibodies. These serologic groups represent the target population for a vaccine that prevents the acquisition of HSV-2, the major pathogen of genital herpes. Methods Patients We analyzed 137 patients who had no evidence of HSV-2 antibodies in their enrollment serum samples. Patients were enrolled in two phase I and two phase II clinical trials (Table 1); 65 patients lacked antibodies to both HSV-1 and HSV-2 at entry (HSV-seronegative), and 72 had only HSV-1 antibodies at entry (HSV-1-seropositive), as determined by HSV Western blot assay [7, 38]. The two initial phase I, open-label trials were done in a dose-escalating format: The first patients received 10-micrograms doses of gD2 and gB2, the second cohort received 30 g of each protein, and the last cohort received 100 g of each protein (Table 1). In the subsequent two phase II trials, patients were randomly assigned to receive one of these three doses. Because the demographic, side-effect, and immunogenicity profiles of the 23 patients enrolled in the dose-escalating trials and the 114 patients enrolled in the randomized trials were similar, the data have been combined and analyzed together. All patients were recruited from three metropolitan areas, were in excellent health without chronic disease or evidence of genital HSV-2 infection, and were not receiving long-term immunosuppressive therapy or acyclovir. The 114 patients enrolled in the phase II trials were selected from 313 volunteers originally screened for the trial, of whom 83 were rejected because they had HSV-2 antibodies. Fifty of the 67 patients seronegative for HSV and 64 of the 163 patients seropositive for HSV-1 were enrolled. Vaccine was administered intramuscularly into the deltoid at 0, 1, and 6 months. Serum samples for HSV-2 antibody assays were obtained before enrollment and at study days 0 (before immunization), 28, 42, 180, 194, 270, and 360. All patients were examined in the clinic for 30 minutes after each injection and again 24 hours after injection. In addition, patients were given diary cards to record local symptoms (pain, erythema, and induration), constitutional symptoms (headache, fever, malaise, and myalgia), and any disruption of activities on a daily basis for the 7 days after each injection. Table 1. Patient Enrollment in Phase I and II Trials of a Recombinant HSV-2 Vaccine Containing Glycoproteins gB2 and gD2* Side effects were classified as mild, moderate, or severe according to the following definitions: mild = transient, with no limitation in activity; moderate = moderate effect on the patients daily activity; and severe = medical intervention required, with a marked effect on patients daily activity. Occurrences of erythema or induration were recorded as 0 to 10 mm, greater than 10 to 30 mm, greater than 30 to 50 mm, and greater than 50 mm on the basis of the maximal diameter measured during the visits or from diary cards for the 7-day observation period after each injection. The maximum severity of each symptom recorded during this 7-day period is similarly presented. Blood was drawn on days 0, 1, and 14 for the assessment of serum chemistries, liver functions, leukocyte counts, and erythrocyte sedimentation rates. A single lot of vaccine was used for each antigen dose for all vaccinations. The protocol was approved by the institutional review board at each institution, and each patient signed an informed consent form for the screening and vaccination protocol regimen. Patients were monetarily compensated for their time and were instructed to avoid sexual exposure to genital lesions and persons with HSV-2 infection. To compare the HSV gB2, gD2, and neutralizing antibody titers in vaccine recipients with those found in naturally infected persons, we analyzed these HSV antibody responses in unimmunized persons whose HSV Western blot results indicated the presence of only HSV-1 antibodies (n = 249) or only HSV-2 antibodies (n = 133). These persons were being screened for participation in other vaccine trials. Ninety-eight (74%) of the 133 persons seropositive for HSV-2 had frequently recurring (4 to 14 episodes per year), culture-proven, symptomatic genital HSV-2 infection [39]. We did not collect information on the symptoms of the remaining 35 patients seropositive for HSV-2 and the 249 patients seropositive for HSV-1. Thus, the naturally infected comparison group represented a cross-section of persons with subclinical and clinical HSV-1 and HSV-2 infection. Because neutralizing antibodies to HSV-2 have not been found to vary with symptomatic reactivations and because the HSV-2 titers of the 98 symptomatic and the 35 other HSV-2-seropositive patients did not differ, we used the initial serum sample that was collected for assaying the gB2, gD2, and HSV neutralizing antibodies among the naturally infected cohort [39, 40]. Laboratory Studies Antibodies to HSV-2 gB and gD were measured by an enzyme-linked immunosorbent assay (ELISA) as previously described [41]. In brief, 50 L of recombinant proteins diluted to 6 g/mL were separately dispensed into 96-well microtiter plates and incubated for 1 hour at 37 C. Unbound antigen was removed by washing. Serum samples were initially diluted 1:27, the lowest dilution that gave no background value in seronegative patients. Serial dilutions that increased threefold from 1:27 to 1:59 049 were made in the plate, and the plates were incubated for 1 hour at 37 C. Unbound antibodies were removed by washing, and bound antibody-antigen complexes were detected by goat antihuman IgG conjugated to horseradish peroxidase, with subsequent colorimetric development with o-phenylenediamine dihydrochloride (Sigma, St. Louis, Missouri) as the substrate. Optical densities were read at 490 nm with a Titertek spectrophotometer (Flow Laboratories, McClean, Virginia), and titers were determined as the reciprocal of the dilution at which an optical density of 1.0 was reached. A standard human HSV-positive serum sample (Boston Biomedica, Inc., West Bridgewater, Massachusetts) was included on each plate, and the final titer was adjusted so that it matched the standard curve. All serum samples were run in duplicate against both antigens, and reported titers represent the average of the two assay points. In addition, a second internal standard was run on each plate to confirm the internal consistency of the assay. The coefficients of variation for the gB2 and gD2 assays were approximately 34% and 30%, respectively. Neutralizing HSV-2 antibody titers were measured with a complement-dependent microneutralization assay. Serum samples were diluted with a twofold serial dilution and then combined with 1.7 102 plaque-forming units of HSV-2 strain 333 [42] and an equal volume of guinea pig complement at a 1:125 dilution (Gibco BRL, Gaithersburg, Maryland) for 2 hours at 37 C. The mixture was subsequently transferred to a 96-well microtiter plate that contained confluent Vero cell mo

Immunotherapy of Recurrent Genital Herpes with Recombinant Herpes Simplex Virus Type 2 Glycoproteins D and B: Results of a Placebo-Controlled Vaccine Trial

To determine the safety, immunogenicity, and efficacy of a recombinant herpes simplex virus type 2 glycoprotein D and B vaccine in the treatment of recurrent genital herpes, a randomized, placebo-controlled trial was held at two referral centers. Healthy patients with 4-14 recurrences per year received injections of both glycoproteins in MF59 adjuvant or of MF59 alone at 0, 2, 12, and 14 months. For 18 study months, the rate and number of recurrences, the duration and severity of the first confirmed recurrence, vaccine immunogenicity, and rates of local and systemic reactions were determined. The monthly rate of recurrences was not significantly improved, but the duration and severity of the first study outbreak was reduced significantly by vaccination. Glycoprotein-specific and neutralizing antibodies were boosted by vaccination for the duration of the study. This vaccine is safe and immunogenic and ameliorated an observed first postvaccination genital recurrence, but it does not reduce recurrence frequency.

Effects of Antigen Dose and Immunization Regimens on Antibody Responses to a Cytomegalovirus Glycoprotein B Subunit Vaccine

The purpose of this phase I study was to evaluate the safety and immunogenicity of 2 doses of cytomegalovirus glycoprotein B (CMV gB)/MF59 vaccine at 3 different immunization schedules. Ninety-five volunteers were randomized to 6 groups. Antibodies to gB represent the majority of the CMV-specific neutralizing response. Three groups received 5 microgram of gB antigen combined with MF59 (a proprietary adjuvant) and 3 groups received a 30-microgram dose at 0, 1, and 2 months; 0, 1, and 4 months; or 0, 1, and 6 months. The vaccine was well tolerated, and there was no significant difference in antibody production between the 2 doses. The vaccine induced highest antibody titers when given at 0, 1, and 6 months. A low dose of CMV gB/MF59 may be the preferred dose for future studies.

Limited Antibody-Dependent Cellular Cytotoxicity Antibody Response Induced by a Herpes Simplex Virus Type 2 Subunit Vaccine

The humoral response to a herpes simplex virus (HSV) type 2 subunit vaccine containing recombinant glycoproteins B (gB2) and D (gD2) was tested in 3 groups of patients. These included HSV-seronegative, HSV-1-seropositive, and HSV-2-seropositive individuals. There were excellent antibody responses, as measured by gB2- and gD2-specific ELISAs and HSV-2 neutralization assays. However, in 2 HSV-2 antibody-dependent cellular cytotoxicity (ADCC) assays, there were relatively low antibody responses, especially among HSV-seronegative individuals. The low ADCC responses may be associated with the poor efficacy of this vaccine observed in clinical trials.

Limited Variability of Glycoprotein Gene Sequences and Neutralizing Targets in Herpes Simplex Virus Type 2 Isolates and Stability on Passage in Cell Culture

Nucleotide sequence analyses of polymerase chain reaction-amplified genes were performed to determine whether adaptation of herpes simplex virus type 2 to replication in cultured cells or in internal organs during neonatal disseminated disease results in selection of variants with altered forms of three glycoproteins (gB, gC, or gD) that influence virus entry into cells. No variations in sequence were noted as a consequence of in vitro passage or replication in different organs. Five viruses from different subjects differed with respect to gB, gC, and gD gene sequences, expressing four distinct forms of gB, three of gC, and two of gD. These differences did not confer resistance to neutralization by guinea pig or human antisera from subjects immunized with recombinant gB or gD vaccines and may not be consequential for vaccine development.

Cervical Antibody Responses to a Herpes Simplex Virus Type 2 Glycoprotein Subunit Vaccine

Effective vaccines against genital herpes simplex virus type 2 (HSV-2) may need to induce genital tract immune responses. To determine local antibody responses to HSV-2 glycoproteins gB2 and gD2 in an intramuscular subunit vaccine, cervical secretions from HSV-seronegative women and HSV-1-seropositive women were tested for IgG and IgA to gB2 and gD2 by enhanced chemiluminescence Western blot. Most (94%) of the seronegative subjects developed cervical IgG to gB2, IgG to gD2, and IgA to gB2; 72% developed IgA to gD2. All HSV-1-seropositive subjects had cervical IgG responses to vaccine gB2 and gD2, 85% had IgA responses to gB2, and 50% had IgA responses to gD2. Responses were more rapid and titers more consistently sustained in the HSV-1-seropositive women. Further, vaccination resulted in cervical IgG and IgA titers comparable to those to HSV-2 gB2 and gD2 in response to recurrent HSV-2 genital infection.

Immunogenicity of a Recombinant Human Cytomegalovirus (CMV) gB Vaccine in Toddlers |[bull]| 745

Background: Effective immunization of young children against CMV may decrease transmission from children to adults and prevent severe sequelae including congenital birth defects. Objective: To evaluate the reactogenicity and immunogenicity of CHIRON CMV gB vaccine in toddlers. Methods: We conducted a Phase I trial in which 18 CMV-seronegative children, 12-35 months of age, received either 20μg of CMV gB/MF59 vaccine, a purified recombinant gB protein produced in Chinese hamster ovary cell culture combined with MF59, oil and water adjuvant, (n=15) or a control Hepatitis A vaccine from SmithKline Beecham (n=3) at 0,1, and 6 months. The study was open-label for the first 6 children, and then double-blinded and randomized. Children were monitored for local and systemic reactions for 7 days following each injection. Serologic tests for CMV anti-gB antibody by ELISA and CMV neutralizing antibodies were obtained pre-immunization, 1 month after dose 2 in 50% of subjects, and 1 month after.dose 3 in all subjects. Results: No pattern of reactogenicity was observed; 5 post-immunization reactions were seen in 4 subjects. The GMTs (95% CI) of antibody to gB by ELISA were: 0 pre-immunization (n=18), 27,457 (13,794-54,653) after dose 2 (n=6), and 98,264(39,896-242,025) after dose 3 (n=5). Neutralizing antibody reciprocal GMTs(95% CI) were: 0 pre-immunization (n=18), 90 (51-158) after dose 2 (n=6), and 638 (415-982) after dose 3 (n=13). After dose 3, all children had titers greater than those observed in naturally infected adults.Conclusion: Antibody responses of toddlers were notably higher than among 149 adults given 3 doses of the same vaccine in other trials(neutralization GMT=133). CHIRON CMV gB vaccine is well tolerated and highly immunogenic in toddlers.Supported by Chiron Vaccines, Emeryville, CA