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

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1 1 Cervical Antibody Responses to a Herpes Simplex Virus Type 2 Glycoprotein Subunit Vaccine Rhoda L. Ashley, Flor-Mari Crisostomo, Michael Doss, Rose E. Sekulovich, Rae Lyn Burke, Mary Shaughnessy, Lawrence Corey, Nayak L. Polissar, and Andria G. M. Langenberg University of Washington and The Mountain-Whisper-Light Statistical Consulting, Seattle, Washington; Chiron Vaccines, Emeryville, California 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. Genital herpes continues to spread, partly because of the Here we report the isotype and kinetics of cervical antibody high proportion of subclinical or unrecognized cases and the development after each of three intramuscular doses of an frequency of viral shedding in the absence of symptoms [1]. HSV-2 subunit vaccine. Eighteen HSV-seronegative women That prior immunity to herpes simplex virus (HSV) gives at and 20 women with prior HSV-1 immunity completed a protoleast partial protection against infection is inferred from obser- col to receive three doses of vaccine. Their responses were vations that genital infection with ú1 strain of HSV-2 is rare compared to determine the effects of prior mucosal priming [2], that acquisition of genital herpes is lower in subjects with (in the subjects with prior HSV-1) on local antibody development prior HSV-1 antibodies than in HSV-naive patients [3], and after parenteral boosting. that vertical transmission of genital HSV-2 to the newborn is infrequent from mothers with established infection [4]. These findings suggest that vaccine-induced immunity could be an Materials and Methods effective means of slowing the spread of genital herpes. Because HSV infections begin at mucosal sites, induction of a Subjects. Women aged years were recruited, enrolled, consistent local antibody response is a desirable characteristic and followed up at the University of Washington Virology Re- search Clinic. Women were not pregnant at entry, agreed to use of any candidate vaccine for HSV. reliable birth control methods throughout the study, and were hu- Previously we characterized cervical antibody responses to man immunodeficiency virus negative. Subjects were tested for first episodes of genital herpes in women. These studies showed the presence of HSV-1 and HSV-2 serum antibodies by Western a predominant IgG response to viral protein antigens similar blot [7]. to those of serum antibodies. However, HSV-specific IgA re- Twenty HSV-seronegative women and 20 HSV-1 seropositive sponses differed qualitatively from cognate serum IgA re- women were enrolled. Further serologic testing by a more sensitive sponses [5]. We also demonstrated local anamnestic antibody technique [8] confirmed that 19 women lacked antibodies to HSV- responses in the cervix when HSV-1 seropositive women acinfected 1 and HSV-2, providing serologic evidence that they had not been quired genital HSV-2 infections [6]. with either HSV subtype. This additional testing revealed that 1 subject who was HSV-seronegative by standard screening assay had a low titer of antibodies to HSV-1. This person, therefore, was included in the HSV-1 seropositive group, giving 19 Received 25 September 1997; revised 22 January HSV-seronegative and 21 HSV-1 seropositive subjects. None of Presented in part: 35th Interscience Conference on Antimicrobial Agents the subjects had histories of genital herpes; 4 of the HSV-1 and Chemotherapy, San Francisco, October 1995 (abstract H6). All subjects gave informed consent under a protocol approved by the Univer- seropositive subjects had a history of oral herpes. sity of Washington Human Subjects Review Committee. Eighteen HSV-seronegative and 20 HSV-1 seropositive sub- Financial support: Chiron Vaccines; NIH (AI-30731). jects completed the vaccine schedule of intramuscular doses at Reprints or correspondence: Dr. Rhoda L. Ashley, University of Washington, days 0, 28, and 180. Fourteen seronegative and 16 HSV-1 sero- Virology Office, Rm. G-815, Box CH-82, Children s Hospital & Medipositive subjects consented to a fourth dose given Ç1 year after cal Center, 4800 Sand Point Way N.E., Seattle, WA entry. The vaccine comprised 30 mg each of HSV-2 recombinant The Journal of Infectious Diseases 1998;178: by the Infectious Diseases Society of America. All rights reserved. glycoproteins gb2 and gd2 in MF59 adjuvant (Chiron Vaccines, /98/ $02.00 Emeryville, CA [9]). Sera drawn at the end of the trial were tested

2 2 Ashley et al. JID 1998;178 (July) by Western blot in parallel with sera drawn at the start of the trial test artifacts that prevented accurate IgA assessment. Of 18 day 0 to detect serologic evidence of HSV-2 infection in all subjects and specimens from subjects that were tested for cervical IgG, 14 of HSV-1 infection in the HSV-seronegative subjects during the (78%) were positive for gd2 and 12 (67%) for gd2. As shown trial. None of the subjects was found to have seroconverted to previously, HSV-1 infection results in detectable cervical antibod- HSV-1 or HSV-2. ies to gb2 and gd2 because of the extensive cross-reactivity of Cervical secretions from 11 subjects who were seronegative for these proteins between HSV-1 and HSV-2 [6]. HSV by enhanced chemiluminescence Western blot (ECL-WB) Titer determination by strip immunoblot assay. Cervical secretions [8] were pooled to use as background control samples in the ECL- were serially diluted 4-fold in 4% goat serum in PBS starting WB test. An additional 6 control subjects were HSV-2 seroposi- at a dilution of 1:20 for IgA and at 1:80 for IgG. Custom nitrocellulose tive with histories of culture-documented recurrent genital herpes strips containing bands of nondenatured recombinant gb2 and lesions. Cervical and vulvar samples from these HSV-2 seroposi- gd2 were provided by Chiron Vaccines. Strips were incubated for tive control subjects were further characterized by HSV polymerase 1 h in 0.5 Tween 20 in PBS, and then used as described [5]. Strips chain reaction as being negative for HSV shedding at the time were exposed to film for 30 s and then for periods ranging from of cervical sampling. 2 s to 2 min. End-point titers were taken as the reciprocal of the Sampling. Cervical samples were collected on days 0, 28, 42, highest dilution showing a clear gb2 or gd2 band at the selected 90, 180, 194, 270, and 360 and 14 days after the fourth dose. As exposure time, as shown for cervical IgG (figure 1A) and cervical described in detail [5], two sequential pairs of tear flow indicator IgA (figure 1B) from 1 HSV-1 seropositive subject. The test was strips were placed in the cervical os until secretions had soaked repeated if titer determinations of two readers were not in agree- up to the strips shoulders. After trimming, the four saturated stubs ment. By comparing end-point titers with those generated by humanized were placed in a vial with 0.5 ml of 0.01% sodium azide in PBS monoclonal antibodies of known concentration (see bewere and frozen at 020 C. Serum was obtained at each clinic visit and low), we estimate that a cervical IgG titer of 1:80 represents Ç78 stored at 020 C. ng/ml IgG to gb and 313 ng/ml IgG to gd. Local antibody testing. Samples were processed as described Humanized IgG monoclonal antibodies against gb2 and gd2 previously [5]; none was positive for blood (Hemoccult; Smithwere (provided by R. Whitley, University of Alabama, Birmingham) Kline Diagnostics, San Jose, CA). used to select films for end-point determination. Monoclonal Samples were tested by ECL-WB using recombinant gb2 and antibodies were serially diluted 4-fold in 4% goat serum in PBS gd2 as the antigens [8]. Because the recombinant proteins con- starting at 1:4000 and reacted with assay strips as described above. tained several species with similar electrophoretic mobilities, gb2 A monoclonal antibody test was run with each subject s secretions. and gd2 were run on separate gels. Twenty microliters of each The exposure time for the film to be used for end-point determina- cervical sample was diluted 1:50 in Blotto and incubated with blots tion was determined by comparing the monoclonal antibody band overnight at room temperature. Blots were washed, and bound intensity at each dilution with that of an external reference standard antibodies were detected with goat anti-human IgA (a chain) or run. Films were selected to be as close as possible in intensity of goat anti-human IgG (g chain) and ECL detection reagents (Amer- these control bands to minimize reading artifact (figure 1C). sham Life Sciences, Oakville, Canada) [5]. Statistical methods. The proportions of seronegative and HSV- Samples from days 0 to 270 (8 samples) from a single subject 1 seropositive subjects having an IgG and IgA response to the were run in the same immunostaining run on blots derived from vaccine glycoproteins were compared by cross-tabulation, with a single gel containing gb2 and a second gel containing gd2. A statistical significance based on x 2 or Fisher s exact test. second set of blots was used for days 360 and 374 to assess IgA values õ1:20 and IgG values õ1:80 were assigned values responses to the fourth dose. Blots were exposed to film for periods of 1:10 and titers were log 10 transformed for these analyses. Geo- from 10 s to 4 min. Film profiles were selected for scoring, using metric mean titers are presented along with their SDs. Because of the maximal film exposure time that gave discrete banding in the presence of outliers or extreme values for some samples, the the lane developed with a cervical secretion control pool from nonparametric Wilcoxon Mann-Whitney test was used to calcu- unvaccinated HSV-2 seropositive subjects. Most profiles were seseronegative late statistical significance of differences between groups (e.g., lected for scoring from 1-min exposures (IgA) or 30-s exposures vs. seropositive.) The Wilcoxon signed ranks test was (IgG). used to compare paired values within a group (e.g., titers before Profiles were scored as positive for vaccine-related response in and after a dose). seronegative subjects if vaccine bands were detectable 28 days The Spearman correlation coefficient was used to determine if after the first vaccine dose and 14 days after subsequent doses serum and cervical IgG responses tended to change together over compared with the profile of cervical secretions from the same time. For each subject, the Spearman correlation coefficient besubject just before that immunization. Profiles were scored as posivation tween paired values (serum, cervical) was calculated for all obsertive for vaccine-related responses in HSV-1 seropositive subjects times. The correlation ranged between 0.0 and {1.0, repre- if vaccine bands appeared de novo or increased in intensity in senting no correlation and perfect correlation (positive or negative), blots of secretions taken 28 days after the first vaccine dose comnull respectively. The Wilcoxon signed ranks test was used to test the pared with the day 0 sample and 14 days after subsequent doses of hypothesis that the mean correlation is zero, that is, that serum vaccine compared with profiles of secretions collected immediately and cervical responses are unrelated. before that immunization. Of 17 HSV-1 seropositive subjects with evaluable specimens Results for cervical IgA at day 0, 14 (82%) had IgA to gb2 and 4 (24%) Cervical IgG and IgA responses to vaccine in seronegative had IgA to gd2. The 18th subject s day 0 specimen resulted in subjects. Paired Western blots of pre- and postvaccine secre-

3 JID 1998;178 (July) Cervical Antibodies to HSV-2 Vaccine 3 Figure 1. Strip immunoblot endpoint titer determination for IgG (A) and IgA (B) to gb2 and gd2 in HSV- 1 seropositive subject receiving HSV-2 gd2-gb2 subunit vaccine. Cervical secretions from day 0, 14, 28, 42, and 90 were serially diluted 4-fold from 1:80 (IgG) or from 1:20 (IgA) and reacted with immunoblots containing gd2 and gb2. C shows results of IgG humanized monoclonal antibody (HuMAb) against gd2 and gb2 4-fold diluted from 1:4000 to 1:1,024,000. Set of blots at left is standard against which films were compared from test runs; set on the right was generated in this run. Film exposure times showing end-point titer of 1:64,000 for gb2 and 1:16,000 for gd2 were used for vaccine antibody titer determinations. Control bands on strips include, from top, high-titer human IgG, mouse monoclonal antibody to human IgM, and human IgG. tions were compared for evidence of responses to gb2 or gd2. increased in intensity at day 42 and was therefore scored as a Appearance of a new band in the gb2 or gd2 location of response. Comparing day 194 and day 180 profiles resulted in the blot or increased band intensity of secretions taken after scoring an IgG response to both gb2 and gd2 given in dose vaccination compared with the day of vaccination was scored 3. This subject demonstrated decreased levels of IgG and IgA as a response to that particular vaccine dose. at day 270, suggesting a waning of titers 3 months after the Cervical antibody responses to the vaccine proteins were third dose. observed in most subjects, but not necessarily to each dose. Of All of this subject s post-dose profiles at day 28, 42, and the 18 seronegative subjects who received three doses, have antibody not detected at day 0, before immunization. (94%) had IgG responses to gb2 in at least 1 pair of samples. However, with IgA in particular, intensity of gb2 bands did For the other antibody responses, 17 pairs could be evaluated. not change dramatically once a response was noted. Responses Sixteen (94%) of 17 seronegative subjects developed IgG to to the fourth dose were determined in paired day 360 and day gd2 and IgA to gb2 after at least one dose; 13 (72%) of secretions analyzed at a later date with a second set of developed IgA responses to gd2 that were detected in at least blots. IgG or IgA responses were not apparent to gb2 or gd2 1 pair of samples. Responses to dose 4 in the 14 seronegative subjects were less prevalent than to earlier doses (table 1). The responses to individual doses 2, 3, and 4 represented Table 1. Prevalence of cervical IgG and IgA responses to HSV-2 first-ever responses in some subjects, recovery of antibody to gd2-gb2 subunit vaccine in seronegative subjects. dose 1 that had been lost before subsequent doses in other No. of responses/total no. of evaluable specimen pairs (%) subjects, or boosting of prior responses as noted by an increased Vaccine band intensity over that of the prior postvaccine sample. Figure dose IgG to gb2 IgG to gd2 IgA to gb2 IgA to gd2 2 illustrates a typical pattern of cervical IgG (figure 2A) and cervical IgA responses (figure 2B) in a seronegative subject. 1 6/16 (38) 6/16 (38) 9/17 (53) 3/17 (18) Comparing day 28 profiles to day 0 profiles revealed the pres- 2 11/17 (65) 13/16 (81) 11/17 (65) 9/17 (53) 3 13/17 (76) 14/17 (83) 9/16 (56) 4/16 (25) ence of de novo IgG to gb2 and gd2 and de novo IgA to gb 4 5/14 (36) 9/14 (64) 5/14 (36) 4/14 (29) and gd. IgA to gd2 also was apparent after the third dose in this subject. Responses to dose 2 were scored by comparing NOTE. Paired specimens from days 0 and 28 (dose 1), from days 28 and day 42 profiles with those from day 28. Intensity of gb2 bands 42 (dose 2), from days 180 and 194 (dose 3), and from days 360 and 374 (dose 4) were subjected to enhanced chemiluminescence Western blot. De actually decreased slightly for both IgG and IgA; thus, no novo appearance of gb2 or gd2 bands or increased intensity of gb2 or gd2 response was scored for the second dose. The IgG gd2 band bands following vaccination was recorded as response.

4 4 Ashley et al. JID 1998;178 (July) mean titers than those observed after the second or third doses (figure 3A). Mean end point titers for specimens taken at days 90 and 180 (between doses 2 and 3) showed progressive waning of antibody titers (figure 3A). Similarly, mean titers dropped by day 360 between the third and fourth doses (figure 3A). Thus, in seronegative subjects, the third and fourth doses did not boost antibody titers above those achieved with two doses but, rather, served to partially or fully restore titers that had waned. Cervical IgG and IgA responses to vaccine in seropositive subjects. All of the 20 seropositive subjects receiving three Figure 2. Cervical antibody profiles in HSV-seronegative subject doses had increased cervical IgG band intensities of gb2 and gd2 after one to three doses. Seventeen (85%) of 20 subjects had increased levels of cervical IgA to gb2 and 10 (50%) developed additional IgA to gd2 after at least one dose. A higher proportion of subjects responded to the first dose than to later doses (table 2). Response patterns differed between seronegative and HSV- 1 seropositive subjects in the following ways: A higher proportion of HSV-1 seropositive than seronegative subjects had responses to the first dose (P õ.01 for IgG to gb2 and gd2). In contrast, a higher proportion of seronegative subjects had receiving 4 doses of HSV gd2-gb2 subunit vaccine. Cervical secretions responses to the second dose (P õ.05 for IgG to gd2; P õ from days 0, 14, 28, 42, 90, 180, 194, and 270 were tested on.01 for IgA to gb2 and gd2). Thus, it appears that seronegative gb2-containing Western blots (left-hand blot of each pair) and gd2- subjects require more antigen presentation events to give evicontaining blots (right-hand blot of each pair) for IgG (A) or IgA dence of vaccine response than do those with prior experience (B). Similar test was done in separate run on cervical secretions from day 360 and 374. Control pools of cervical secretions from with HSV-1. Seronegative subjects were also more likely to seronegative unvaccinated subjects (0) and from HSV-2 infected respond to the third dose (P õ.05 for IgG to gd2). subjects (/) were tested in parallel; controls for first run (day 0 Magnitude and duration of cervical IgG and IgA responses 270) are not shown. Note 2 species of gb2. Responses were scored in seropositive subjects. End-point titers established using based only on major, more slowly migrating form. Y, N indicate strip immunoblots are illustrated for 1 HSV-1 seropositive whether band intensity increased (Y) or remained equivalent or desubject for cervical IgG (figure 1A) and IgA (figure 1B) in creased (N) compared with profile obtained before vaccine dose. specimens collected on days 0, 14, 28, 42, and 90. In this subject, IgG and IgA were present to both gb2 and gd2 before in the subject illustrated in figure 2; however, IgG to gb2 and vaccination (day 0). Both IgG and IgA titers increased by day gd2 and IgA to gb2 are clearly shown compared with day IgA titers waned; day 42 titers did not recover to day 14 preimmunization profiles. levels after a second dose. Similarly, IgG titers at day 42 did Magnitude and duration of cervical IgG and IgA responses not reflect a response to the second dose given at day 28. in seronegative subjects. Examination of blots such as those In the seropositive subjects, geometric mean IgG titers rose in figure 2 revealed that in many cases, responses to doses 3 after dose 1 and remained quite constant thereafter (figure 3B). or 4 did not dramatically increase the band intensity seen after Mean titers of IgA in seropositive subjects rose after the first dose 1 or 2. It appeared that doses beyond 2 in seronegative dose and remained relatively constant except for slight titer subjects served more to restore waning levels of antibodies decreases before the third dose (day 180) and before the fourth than to boost antibody levels beyond those derived from prior dose (day 360). Thus, HSV-1 seropositive subjects had less doses. However, since observation of blot profiles is not quantitative, dramatic fluctuations in titers over the course of the vaccination we attempted to confirm this impression by determining protocol than did seronegative subjects. end-point titers for the subset of seronegative subjects with Titers of cervical antibodies to vaccine versus natural infec- sufficient residual samples. tion. Western blots from this study had gb2 and gd2 band Increases in mean log 10 titers were observed for both IgG intensities that were similar to or more intense than those proand IgA against both proteins for all doses (figure 3A). Mean duced by cervical specimens from patients with genital herpes, titers were higher at the end of the trial (after three doses) than suggesting that vaccine responses were comparable in titer with after the first dose for IgG and IgA to both gb2 and gd2. Titers those elicited by mucosal infection. To test this hypothesis, after three doses were equal to or slightly lower than after the end-point titers after three doses (day 194) were compared with second dose. The fourth dose did not result in higher geometric those obtained from women with culture-documented recurrent

5 JID 1998;178 (July) Cervical Antibodies to HSV-2 Vaccine 5 Figure 3. Geometric mean titers in HSV-seronegative (A) and HSV-1 seropositive subjects (B) receiving 3 or 4 doses of HSV-2 gd2-gb2 subunit vaccine. Titers of IgG to gb2, IgG to gd2, IgA to gb2, and IgA to gd2 were measured by end-point dilution. Geometric mean titers were plotted to demonstrate fluctuations in responses with time and with additional doses of vaccine. Bars denote 1 SE. No. of subjects varied from 6 to 11 per time point for seronegative and 11 to 19 for seropositive subjects, as noted at base of figure. HSV-2 infections. The HSV-2 infected subjects were sampled than in either the seronegative vaccine recipients (P õ.01 for for cervical antibodies and, at the same visit, by polymerase IgG to gb2 and P õ.05 for IgG to gd2) or the HSV-2 chain reaction testing to detect HSV DNA. Only samples taken infected subjects (P õ.01 for IgG to gb2 and gd2). There at times when HSV DNA was not detected were used in this was a trend toward higher IgA titers in HSV-1 seropositive comparison to avoid bias introduced by local antigen-antibody vaccine recipients than in either seronegative or naturally infected complexes, which would not be detected by the strip immunoblot subjects. IgG and IgA titers of the infected subjects were system. Titers from 8 seronegative vaccine recipients, somewhat higher than those of seronegative vaccine subjects, from 18 HSV-1 seropositive vaccine recipients, and from 9 but the differences were not statistically significant. naturally HSV-2 infected subjects were compared (table 3). Comparison of cervical and serum IgG titers. This study Mean titers of IgG and IgA to gb2 and gd2 were significantly offered the unique opportunity to explore the relationship beanti- higher in the HSV-1 seropositive vaccine recipients tween serum responses and the development of cervical

6 6 Ashley et al. JID 1998;178 (July) Table 2. Prevalence of cervical IgG and IgA responses to HSV-2 vaccine glycoprotein components in virtually all human subgb-gd subunit vaccine in HSV-seropositive subjects. jects tested. As we found with natural HSV-2 genital herpes [5, 6], IgG was the predominant cervical antibody isotype in- No. of responses/total no. of evaluable specimen pairs (%) Vaccine duced by the subunit vaccine. Total IgG levels in the female dose IgG to gb IgG to gd IgA to gb IgA to gd genital tract usually exceed those of IgA [10, 11]. Furthermore, IgG has been shown to be the major genital tract isotype in 1 20/20* (100) 17/20* (85) 12/19 (63) 10/19 (53) response to human immunodeficiency virus [12] and to intra- 2 6/19 (32) 8/19 (42) 3/19* (16) 1/19* (5) muscular immunization with tetanus toxoid [13]. 3 10/20 (50) 9/20 (45) 6/19 (32) 3/19 (16) 4 9/16 (56) 6/16 (38) 9/16 (56) 1/16 (6) The source of cervical IgG may be, in part, local plasma cells [14, 15], or the IgG may be derived from serum pools, NOTE. Paired specimens from days 0 and 28 (dose 1), from days 28 and either by passive diffusion [16] or by an active transport mecha- 42 (dose 2), from days 180 and 194 (dose 3), and from days 360 and 374 nism [11, 13]. The finding that fluctuations in cervical IgG (dose 4) were subjected to enhanced chemiluminescence Western blot. Intiters were significantly correlated with those of serum IgG is creased intensity of gb2 or gd2 band was recorded as response. * P õ.01 vs. seronegative subjects. consistent with the notion that most of the cervical IgG may P õ.05 vs. seronegative subjects. be derived from the serum compartment after immunization with this gd2-gb2-mf59 subunit vaccine. A similar correlation bodies after vaccination. From 12 seronegative subjects, 2 8 between serum and parotid or nasal IgG to a cytomegalovirus cervical IgG values per subject (median, 6) were available to subunit vaccine in MF59 adjuvant has been reported [17], sug- compare with gb2- and gd2-specific EIA titers from sera gesting that this adjuvant may affect the induction and delivery drawn at the same visit; from 19 seropositive subjects, 4 8 mechanisms for IgG in oral and respiratory as well as genital (median, 8) values per subject were available. secretions. A more direct method to infer serum antibody tran- Serum and cervical responses over time showed a moderate sudation has been described but was not applied in these studies but highly significant mean correlation in both HSV-1 sero- [18]. positive and HSV-seronegative subjects. In each group, correla- Passive immunization with serum IgG has been shown to tions of serum and cervical IgG to gb2 and correlations of be protective against vaginal challenge with HSV in mice [19]. serum and cervical IgG to gd2 were about equal. The Spearman It has been suggested that in humans, threshold serum IgG correlation for serum and cervical IgG to gb2 in the seronega- levels induced by vaccines against mucosal pathogens are most tive group was.72 {.27 (mean { SD) with 12 patients, yield- closely correlated with protection [20]. Additional study will ing P Å.002, to reject the null hypothesis of zero correlation. be needed to determine the function of the IgG identified in For serum and cervical IgG to gd2, the correlation was very this study. similar: n Å 11, r Å.79 {.20, P Å.003. In the seropositive Cervical IgA to the subunit vaccine was less prevalent than group, the correlations between serum and cervical titers were IgG and lower in titer than cervical IgG. The methods used somewhat weaker but still significant: for IgG to gb2, n Å 18, for this study did not identify different molecular forms of IgA r Å.39 {.24, P õ.001; for IgG to gd2, n Å 19, r Å.36 {.42, (dimeric versus monomeric), nor were we able to determine if P Å.004. These correlations indicate that serum and cervical the IgA detected contained secretory component, a covalently antibody titers changed, in concert, over time. bound moiety obtained via active transport and maturation of dimeric IgA into the mucosal lumen. Because the serum EIA Discussion did not measure IgA, we could not determine if the fluctuations We report here that intramuscular injections of an HSV-2 in IgA titers reflected serum IgA fluctuations. Thus, while IgA subunit vaccine result in cervical IgG and IgA to both of the appears to constitute a substantial minority of the immunoglob- Table 3. Comparison of cervical IgG and IgA geometric mean titers in subjects who received 3 doses of vaccine with those from subjects with genital HSV-2 infection. IgG IgA Group gb2 gd2 gb2 gd2 Seronegative vaccine recipients 2.7 { 1.2* (n Å 8) 2.9 { 1.4 (n Å 7) 1.9 { 0.7 (n Å 6) 2.2 { 1.1 (n Å 8) Seropositive vaccine recipients 3.9 { 0.4* (n Å 18) 3.9 { 0.4 (n Å 18) 2.7 { 0.8 (n Å 18) 2.7 { 0.8 (n Å 18) Subjects with HSV-2 infection 3.2 { 0.6 (n Å 9) 3.3 { 0.6 (n Å 9) 2.4 { 0.7 (n Å 9) 2.4 { 1.2 (n Å 9) NOTE. Data are geometric mean cervical log 10 end-point titers ({ SD). * P õ.01 between seronegative and seropositive groups. P õ.05 between seronegative and seropositive groups. P õ.01 between seropositive and infected groups.

7 JID 1998;178 (July) Cervical Antibodies to HSV-2 Vaccine 7 ulins that arise in response to the HSV subunit vaccine, its 3. Mertz GJ, Benedetti J, Ashley R, Selke S, Corey L. Risk factors for sexual transmission of genital herpes. Ann Intern Med 1992;116: characteristics and possible source are unclear. 4. Brown ZA, Selke S, Zeh J, et al. The acquisition of herpes simplex virus Previous studies with this HSV-2 vaccine revealed equivaduring pregnancy. N Engl J Med 1997;337: lent or higher serum antibody titers to vaccine than to natural 5. Ashley RL, Corey L, Dalessio J, et al. Protein-specific cervical antibody HSV-2 infection [9]. Assuming local antibody functions either responses to primary genital herpes simplex virus type 2 infections. J protect against HSV-2 superinfection or are markers of other Infect Dis 1994;170: Ashley R, Wald A, Corey L. Cervical antibodies in patients with oral protective local factors, cervical antibody titers in natural infecherpes simplex virus type 1 (HSV-1) infection: local anamnestic retions provide a benchmark for potentially protective levels of sponse after genital HSV-2 infection. J Virol 1994;68: antibodies induced by vaccine. Mean cervical antibody titers 7. Ashley RL, Militoni J, Lee F, Nahmias A, Corey L. Comparison of Western after three doses of vaccine in the seropositive group approxiassay blot (immunoblot) and glycoprotein G specific immunodot enzyme mated (IgA) or significantly exceeded (IgG) those seen with for detecting antibodies to herpes simplex virus types 1 and 2 in human sera. J Clin Microbiol 1988;26: natural HSV-2 infection. The seronegative group had lower 8. Dalessio J, Ashley R. Highly sensitive enhanced chemiluminescence immean antibody titers than did either the seropositive vaccine munodetection method for herpes simplex virus type 2 Western immugroup or the group with natural HSV-2 infection. noblot. J Clin Microbiol 1992;30: Seropositive subjects responded to the first dose with titers 9. Langenberg AGM, Burke RL, Adair SF, et al. A recombinant glycoprotein that remained high over the course of the study. Seronegative vaccine for herpes simplex type 2: safety and immunogenicity. Ann Intern Med 1995;122: subjects required two doses for maximal titers and had sharper 10. Cerasaro M, Nicotra M, Coghi IM, Stampone L, Amodei M, Dondero F. declines in titer between doses. These differences in kinetics Detection of antispermatozoal iso-antibodies in cervical mucus from can most logically be ascribed to prior mucosal stimulation infertile and fertile women using the Shulman-Hekman capillary test. with antigen via oral HSV-1 infection in the group with prior Immunol Factors Hum Reprod 1982;51 7. HSV-1 antibodies. Cervical IgG and IgA to HSV are present 11. Hocini H, Barra A, Belec L, et al. Systemic and secretory humoral immunity in the normal human vaginal tract. Scand J Immunol 1995;42:269 in varying levels in subjects with prior HSV-1 infection [6]. 74. The relatively flat mean titer curve of the seropositive subjects 12. Lu XS, Belec L, Pillot J. Anti-gp160 IgG and IgA antibodies associated over four doses of vaccine and the fact that many did not with a large increase in total IgG in cervicovaginal secretions from increase their titers after doses 3 or 4 suggest that an upper limit human immunodeficiency virus type 1 infected women. J Infect Dis of responsiveness was reached beyond which further antigen 1993;167: Bouvet JP, Belec L, Pires R, Pillot J. Immunoglobulin G antibodies in presentation failed to elicit a heightened response. human vaginal secretions after parenteral vaccination. Infect Immun The functional utility of the genital tract antibodies that we 1994;62: have identified to a systematically delivered subunit HSV vac- 14. Bjercke S, Brandtzaeg P. Glandular distribution of immunoglobulins, J cine is not known. An efficacious vaccine for prevention of chain, secretory component, and HLA-DR in the human endometrium genital HSV-2 is likely to be associated with meaningful elicita- throughout the menstrual cycle. Hum Reprod 1993;8: Kutteh WH, Hatch KD, Blackwell RE, Mestecky J. Secretory immune tion of genital tract immune responses. system of the female reproductive tract. I. Immunoglobulin and secretory component containing cells. Obstet Gynecol 1988;71: Acknowledgments 16. Wagner DK, Clements ML, Reimer CB, Snyder MH, Nelson DL, Murphy BR. Analysis of IgG antibody responses after live and inactivated influenza We gratefully acknowledge clinical research associates Carolyn A vaccine indicate that nasal wash IgG is a transudate from serum. Gee and Astrid Ramens for clinical oversight and protocol manage- J Clin Microbiol 1987;25: ment; Anna Wald, Medical Director at the Virology Research 17. Wang JB, Adler SP, Hempfling S, et al. Mucosal antibodies to human cytomegalovirus glycoprotein B occur following both natural infection Clinic, and Cornelia Dekker for programmatic support; David Kerr and immunization with human cytomegalovirus. J Infect Dis 1996;174: for excellent statistical computing; Julie Dalessio for expert techni cal advice; and Sharon Risley for assistance with the manuscript. 18. Bélec L, Dupré T, Prazuck T, et al. Cervicovaginal overproduction of specific IgG to human immunodeficiency virus (HIV) contrasts with References normal or impaired IgA local responses in HIV infection. J Infect Dis 1995;172: Corey L, Wald A. New developments in the biology of genital herpes. In: 19. Whaley KJ, Zeitlin L, Barratt RA, Hoen TE, Cone RA. Passive immuniza- Sacks SL, Straus SE, Whitley RJ, Griffiths PD, eds. Clinical manageherpes tion of the vagina protects mice against vaginal transmission of genital ment of herpes viruses. Washington, DC: IOS Press, 1995: infections. J Infect Dis 1994;169: Schmidt OW, Fife KH, Corey L. Reinfection is an uncommon occurrence 20. Robbins JB, Schneerson R, Szu SC. Perspective: hypothesis: serum IgG in patients with symptomatic recurrent genital herpes. J Infect Dis 1984; antibody is sufficient to confer protection against infectious diseases by 149: inactivating the inoculum. J Infect Dis 1995;171: