CD4+ T cells provide intermolecular help to generate robust antibody responses in vaccinia virus-vaccinated humans.

Immunization with vaccinia virus elicits a protective Ab response that is almost completely CD4(+) T cell dependent. A recent study in a rodent model observed a deterministic linkage between Ab and CD4(+) T cell responses to particular vaccinia virus proteins suggesting that CD4(+) T cell help is preferentially provided to B cells with the same protein specificity (Sette et al. 2008. Immunity 28: 847-858). However, a causal linkage between Ab and CD4(+) T cell responses to vaccinia or any other large pathogen in humans has yet to be done. In this study, we measured the Ab and CD4(+) T cell responses against four vaccinia viral proteins (A27L, A33R, B5R, and L1R) known to be strongly targeted by humoral and cellular responses induced by vaccinia virus vaccination in 90 recently vaccinated and 7 long-term vaccinia-immunized human donors. Our data indicate that there is no direct linkage between Ab and CD4(+) T cell responses against each individual protein in both short-term and long-term immunized donors. Together with the observation that the presence of immune responses to these four proteins is linked together within donors, our data suggest that in vaccinia-immunized humans, individual viral proteins are not the primary recognition unit of CD4(+) T cell help for B cells. Therefore, we have for the first time, to our knowledge, shown evidence that CD4(+) T cells provide intermolecular (also known as noncognate or heterotypic) help to generate robust Ab responses against four vaccinia viral proteins in humans.

Age-associated decrease in TLR function in primary human dendritic cells predicts influenza vaccine response.

We evaluated TLR function in primary human dendritic cells (DCs) from 104 young (age 21-30 y) and older (> or =65 y) individuals. We used multicolor flow cytometry and intracellular cytokine staining of myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) and found substantial decreases in older compared with young individuals in TNF-alpha, IL-6, and/or IL-12 (p40) production in mDCs and in TNF-alpha and IFN-alpha production in pDCs in response to TLR1/2, TLR2/6, TLR3, TLR5, and TLR8 engagement in mDCs and TLR7 and TLR9 in pDCs. These differences were highly significant after adjustment for heterogeneity between young and older groups (e.g., gender, race, body mass index, number of comorbid medical conditions) using mixed-effect statistical modeling. Studies of surface and intracellular expression of TLR proteins and of TLR gene expression in purified mDCs and pDCs revealed potential contributions for both transcriptional and posttranscriptional mechanisms in these age-associated effects. Moreover, intracellular cytokine production in the absence of TLR ligand stimulation was elevated in cells from older compared with young individuals, suggesting a dysregulation of cytokine production that may limit further activation by TLR engagement. Our results provide evidence for immunosenescence in DCs; notably, defects in cytokine production were strongly associated with poor Ab response to influenza immunization, a functional consequence of impaired TLR function in the aging innate immune response.

Antibody profiling by proteome microarray reveals the immunogenicity of the attenuated smallpox vaccine modified vaccinia virus ankara is comparable to that of Dryvax.

Modified vaccinia virus Ankara (MVA) is a highly attenuated vaccinia virus that is under consideration as an alternative to the conventional smallpox vaccine Dryvax. MVA was attenuated by extensive passage of vaccinia virus Ankara in chicken embryo fibroblasts. Several immunomodulatory genes and genes that influence host range are deleted or mutated, and replication is aborted in the late stage of infection in most nonavian cells. The effect of these mutations on immunogenicity is not well understood. Since the structural genes appear to be intact in MVA, it is hypothesized that critical targets for antibody neutralization have been retained. To test this, we probed microarrays of the Western Reserve (WR) proteome with sera from humans and macaques after MVA and Dryvax vaccination. As most protein sequences of MVA are 97 to 99% identical to those of other vaccinia virus strains, extensive binding cross-reactivity is expected, except for those deleted or truncated. Despite different hosts and immunization regimens, the MVA and Dryvax antibody profiles were broadly similar, with antibodies against membrane and core proteins being the best conserved. The responses to nonstructural proteins were less well conserved, although these are not expected to influence virus neutralization. The broadest antibody response was obtained for hyperimmune rabbits with WR, which is pathogenic in rabbits. These data indicate that, despite the mutations and deletions in MVA, its overall immunogenicity is broadly comparable to that of Dryvax, particularly at the level of antibodies to membrane proteins. The work supports other information suggesting that MVA may be a useful alternative to Dryvax.

Serum antibody responses after intradermal vaccination against influenza.

BACKGROUND: If found to be safe and immunogenic, reduced doses of influenza vaccine given by the intradermal route could increase the number of available doses of vaccine. METHODS: In an open-label study, we randomly assigned 119 subjects to receive an intradermal injection of trivalent inactivated influenza vaccine, containing 6 mug of hemagglutinin for each antigen (40 percent of the usual dose), and 119 to receive an intramuscular injection of the standard dose of 15 mug of hemagglutinin for each antigen. The two groups were subdivided according to age (18 to 60 years and older than 60 years). RESULTS: Among subjects who were 18 to 60 years of age, serum antibody responses were vigorous and did not differ significantly between the intradermal and intramuscular groups, and all subjects had hemagglutination-inhibition (HAI) titers of at least 1:40. Although the subjects who were older than 60 years of age also had a vigorous antibody response, there was a trend toward a better response in the intramuscular route, but this finding was significant only for antigen to the H3N2 strain. Nevertheless, 100 percent of older subjects in the intramuscular group and 93 percent of such subjects in the intradermal group had an HAI antibody titer to the H3N2 strain of more than 1:40, and 100 percent in each group had a titer of this level for both the H1N1 and B strains. Local pain was significantly more common in the intramuscular group than in the intradermal group among subjects who were 18 to 60 years of age but not among subjects who were over 60 years old. Signs of local inflammation were significantly more common among subjects in the intradermal group than among those in the intramuscular group, in both age groups. CONCLUSIONS: As compared with an intramuscular injection of full-dose influenza vaccine, an intradermal injection of a reduced dose resulted in similarly vigorous antibody responses among persons 18 to 60 years of age but not among those over the age of 60 years.

Clinical responses to undiluted and diluted smallpox vaccine.

BACKGROUND: To evaluate the potential to increase the supply of smallpox vaccine (vaccinia virus), we compared the response to vaccination with 10(8.1), 10(7.2), and 10(7.0) plaque-forming units (pfu) of vaccinia virus per milliliter. METHODS: In this randomized, single-blind, prospective study, 680 adults who had not been previously immunized were inoculated intradermally with undiluted vaccine (mean titer, 10(8.1) pfu per milliliter), a 1:5 dilution, or a 1:10 dilution of vaccinia virus with use of a bifurcated needle, and the site was covered with a semipermeable dressing. Subjects were monitored for vesicle formation (an indicator of the success of vaccination) and adverse events for 56 days after immunization. RESULTS: Success rates did not differ significantly among the groups and ranged from 97.1 to 99.1 percent after the first vaccination. Both the undiluted and diluted vaccines were reactogenic. In addition to the formation of pustules, common adverse events included the formation of satellite lesions, regional lymphadenopathy, fever, headache, nausea, muscle aches, fatigue, and chills consistent with the presence of an acute viral illness. Generalized and localized rashes, including two cases of erythema multiforme, were also observed. CONCLUSIONS: When given by a bifurcated needle, vaccinia virus vaccine can be diluted to a titer as low as 10(7.0) pfu per milliliter (approximately 10,000 pfu per dose) and induce local viral replication and vesicle formation in more than 97 percent of persons.

Dose-related effects of smallpox vaccine.

BACKGROUND: We conducted a double-blind, randomized trial of three dilutions of vaccinia virus vaccine in previously unimmunized adults in order to assess the clinical success rates, humoral responses, and virus-specific activity of cytotoxic T cells and interferon-gamma-producing T cells. METHODS: Sixty healthy adults were inoculated intradermally by bifurcated needle with undiluted vaccine (dose, 10(7.8) plaque-forming units [pfu] per milliliter), a 1:10 dilution (dose, 10(6.5) pfu per milliliter), or a 1:100 dilution (dose, 10(5.0) pfu per milliliter); there were 20 subjects in each group. The subjects were monitored with respect to vesicle formation (an indicator of successful vaccination), the viral titer at the time of peak lesion formation, antiviral antibodies, and cellular immune responses. RESULTS: A vaccinia vesicle developed in 19 of the 20 subjects who received undiluted vaccine (95 percent), 14 of the 20 who received the 1:10 dilution (70 percent), and 3 of the 20 who received the 1:100 dilution (15 percent). One month after vaccination, 34 of 36 subjects with vesicles had antibody responses, as compared with only 1 of 24 subjects without clinical evidence of vaccinia virus replication. Vigorous cytotoxic T-cell and interferon-gamma responses occurred in 94 percent of subjects with vesicles, and a cytotoxic T-cell response occurred in only one subject without a vesicle. CONCLUSIONS: The vaccinia virus vaccine (which was produced in 1982 or earlier) still has substantial potency when administered by a bifurcated needle to previously unvaccinated adults. Diluting the vaccine reduces the rate of successful vaccination. The development of vesicular skin lesions after vaccination correlates with the induction of the antibody and T-cell responses that are considered essential for clearing vaccinia virus infections.

Response to smallpox vaccine in persons immunized in the distant past.

CONTEXT: There is renewed interest in use of smallpox vaccine due to the potential for a bioterrorist attack. This would involve vaccinating health care workers who were previously vaccinated. OBJECTIVE: To evaluate the use of diluted vaccinia virus in vaccination of previously vaccinated (non-naive) participants. DESIGN, SETTING, AND PARTICIPANTS: Eighty non-naive participants, aged 32 to 60 years, were randomized in a single-blinded study to receive either undiluted or diluted (1:3.2, 1:10, or 1:32) doses of smallpox vaccine. A comparison group, aged 18 to 31 years, of 10 vaccinia-naive participants received undiluted vaccine. Participants were enrolled between April 1 and May 15, 2002, at the National Institute of Allergy and Infectious Diseases Vaccine and Treatment Evaluation Unit at Saint Louis University, St Louis, Mo. INTERVENTION: Smallpox vaccine was administered by scarification using 15 skin punctures in the deltoid region of the arm. MAIN OUTCOME MEASURES: Presence of a major reaction, defined as a vesicular or pustular lesion or area of palpable induration surrounding a central lesion following vaccination, and measures of viral shedding and antibody titers. RESULTS: Initial vaccination resulted in a major reaction in 64 of 80 non-naive participants. Ninety-five percent of non-naive participants had major reactions in the undiluted group, 90% in the 1:3.2 dilution group, 81% in the 1:10 dilution group, and 52.6% in the 1:32 dilution group. All (n = 10) of the vaccinia-naive participants had major reactions. Compared with vaccinia-naive participants, non-naive participants had significantly smaller skin lesions (P =.04) and significantly less incidence of fever (P =.02). Preexisting antibody was present in 76 of 80 non-naive participants. Antibody responses were significantly higher and occurred more rapidly in the non-naive participants compared with the vaccinia-naive participants (P =.002 for day 28 and P =.003 for 6 months). Vaccinia-naive participants shed virus from the vaccination site 2 to 6 days longer and had significantly higher peak mean viral titers when compared with the non-naive participants (P =.002). CONCLUSIONS: Previously vaccinated persons can be successfully revaccinated with diluted (

Safety and immunogenicity of IMVAMUNE® smallpox vaccine using different strategies for a post event scenario.

INTRODUCTION: Reintroduction of Variola major as an agent of bioterrorism remains a concern. A shortened dosing schedule of Bavarian Nordic's (BN) IMVAMUNE(®) (modified vaccinia Ankara vaccine against smallpox) was compared to the currently recommended 0- and 28-day schedule for non-inferiority by evaluating the magnitude and kinetics of the immune responses. METHODS: Subjects were assigned to receive IMVAMUNE or placebo administered subcutaneously on Days 0 and 7, Days 0 and 28, or Day 0. Blood was collected for antibody and cell-mediated immune assays. Subjects were followed for safety for 12 months after last vaccination. RESULTS: The primary endpoint of this study was the geometric mean antibody titers (GMT) at 14 days post last vaccination. Of 208 subjects enrolled, 191 received vaccine (Group: 0+7, Group: 0+28 and Group: 0) and 17 received placebo. Moderate/severe systemic reactogenicity after any vaccination were reported by 31.1%, 25.4%, and 28.6% of the subjects for Group: 0+7, Group: 0+28, and Group: 0, respectively (Chi-square test, P=0.77). Based on BN's Plaque Reduction Assay GMTs, Group: 0+7 was non-inferior to Group: 0+28 at Day 4, 180, and 365 after the second vaccination. On Day 14, Group: 0+7 and Group: 0+28 GMT were 10.8 (CI: 9.0, 12.9) and 30.2 (CI: 22.1, 41.1), respectively. Based on BN's Enzyme-linked immunosorbent assay, the proportion of subjects with positive titers for Group: 0+28 was significantly greater than that for Group: 0+7 after second vaccination at Days 4 and 180. By Day 14 after the second dose, the IFN-γ enzyme-linked immunosorbent spot (ELISPOT) responses were similar for Group: 0+28 and Group: 0+7. CONCLUSION: Overall, a standard dose of IMVAMUNE (0.5 mL of 1 x 10(8) TCID/mL) administered subcutaneously was safe and well tolerated. A second dose of IMVAMUNE at Day 28 compared to Day 7 provided greater antibody responses and the maximal number of responders. By Day 14 after the second dose, IFN-γ ELISPOT responses were similar for Group: 0+28 and Group: 0+7.

Analysis of variola and vaccinia virus neutralization assays for smallpox vaccines.

Possible smallpox reemergence drives research for third-generation vaccines that effectively neutralize variola virus. A comparison of neutralization assays using different substrates, variola and vaccinia (Dryvax and modified vaccinia Ankara [MVA]), showed significantly different 90% neutralization titers; Dryvax underestimated while MVA overestimated variola neutralization. Third-generation vaccines may rely upon neutralization as a correlate of protection.

Comparison of the safety and immunogenicity of ACAM1000, ACAM2000 and Dryvax in healthy vaccinia-naive adults.

Currently, more than half of the world's population has no immunity against smallpox variola major virus. This phase I double-blind, randomized trial was conducted to compare the safety and immunogenicity of two clonally derived, cell-culture manufactured vaccinia strains, ACAM1000 and ACAM2000, to the parent vaccine, Dryvax. Thirty vaccinia-naïve subjects were enrolled into each of three groups and vaccines were administered percutaneously using a bifurcated needle at a dose of 1.0x10(8)PFU/mL. All subjects had a primary skin reaction indicating a successful vaccination. The adverse events, 4-fold neutralizing antibody rise and T cell immune responses were similar between the groups.

Comparative immunogenicity of trivalent influenza vaccine administered by intradermal or intramuscular route in healthy adults.

The present study was undertaken with controls using equal doses ID and IM plus the standard full dose IM to assess the role of route of vaccine in immunogenicity of inactivated influenza vaccine. The study was a prospective, randomized, active-controlled, open label clinical trial conducted in healthy young adult outpatients to compare the effect of route (IM versus ID) on antibody responses to influenza vaccine. Volunteers received 3, 6 or 9 microg of vaccine by ID or IM route; 15 microg IM was also studied. Low doses of vaccine given by either route were almost as immunogenic as the standard 15 microg IM dose of influenza vaccine. ID route was not superior to IM vaccine at inducing antibodies. ID vaccine induced significantly more local inflammatory response than IM vaccine.

Prevaccine determination of the expression of costimulatory B7 molecules in activated monocytes predicts influenza vaccine responses in young and older adults.

BACKGROUND: Innate immunity, including Toll-like receptor (TLR)-mediated expression of the B7 costimulatory molecules CD80 and CD86, is critical for vaccine immunity. We examined whether CD80 and CD86 expression vary with aging and predict response to the trivalent inactivated influenza vaccine. METHODS: One hundred sixty-two subjects between 21 and 30 years of age (the young group) or > or =65 years of age (the older group) enrolled before vaccination. We determined TLR-induced monocyte CD80/CD86 expression by flow cytometry and vaccine antibody responses by hemagglutination inhibition. RESULTS: The mean increase in TLR-induced CD80(+) monocytes was reduced in older, compared with young, adults by 68% (P=.0002), and each decile increase of CD80(+) cells was associated with an 8.5% increase in mean number of vaccine strains with a > or =4-fold titer increase (P=.01) and a 3.8% increase in mean number of strains with a postvaccine titer > or =1 : 64 (P=.037). Each decile decrease of CD86(+) cells was associated with an 11% increase in the mean number of strains with a 4-fold increase (P=.002) and a 3.9% increase in the mean number of strains with a postvaccine titer > or =1 : 64 (P=.07). CONCLUSIONS: CD80 and CD86 expression on activated monocytes is highly associated with influenza vaccine response. This approach prospectively identifies adults unlikely to respond to immunization who may benefit from alternative vaccines or antiviral prophylaxis during influenza outbreaks.

Clinical and immunologic responses to multiple doses of IMVAMUNE (Modified Vaccinia Ankara) followed by Dryvax challenge.

Smallpox vaccination with replication deficient vaccinia strains such as Modified Vaccinia Ankara (MVA) may induce protective immunity with improved safety and tolerability profiles compared with currently available smallpox vaccines. Ninety subjects were randomized equally to six groups in a partially blinded, randomized, controlled clinical trial. IMVAMUNE (MVA-BN, Bavarian Nordic A/S, Kvistgård, Denmark) vaccine or placebo was administered at Study Days 0 and 28 by subcutaneous or intramuscular injection and five groups were challenged with Dryvax at study Day 112. Vaccination with two doses of IMVAMUNE was safe and well tolerated compared to Dryvax. IMVAMUNE produced comparable cellular and humoral immune responses to one dose of Dryvax and the immunity induced appears robust 90 days post-vaccination by evidence of attenuated primary cutaneous reaction responses following Dryvax. IMVAMUNE vaccination prior to Dryvax reduced virus replication at the Dryvax site, decreased the size of the primary cutaneous lesion, and decreased the time to healing but did not completely ameliorate the immune response.

Antibody responses to vaccinia membrane proteins after smallpox vaccination.

BACKGROUND: Vaccinia virus (VV) membrane proteins are candidates for orthopoxvirus subunit vaccines and potential targets for therapeutic antibodies. Human antibody responses to these proteins after VV vaccination have not been well characterized. METHODS: Pre- and postvaccination (day 26-30) serum specimens from 80 VV vaccine recipients were examined for immunoglobulin G antibodies specific for B5, A33, A27, and L1 by enzyme-linked immunosorbent assay (ELISA). Responses were compared between vaccinia-naive and previously vaccinated (nonnaive) recipients and between nonnaive recipients of undiluted or 1 : 10 diluted vaccine. RESULTS: VV vaccination elicited anti-A33 and anti-A27 antibodies in nearly all vaccinia-naive subjects (100% and 93%, respectively). Preexisting antibodies were commonly detected in nonnaive subjects (for anti-B5, 68%; for anti-A33, 59%; for anti-A27, 38%; and for anti-L1, 10%). Anti-B5 antibodies were strongly boosted by undiluted vaccine (geometric mean titer [GMT], 151 vs. 1010 for pre- vs. postvaccination; P<.001), whereas anti-L1 antibody responses were less robust (detection rate, 31%; GMT, 75) in nonnaive subjects. Diluted vaccine elicited antibody responses that were similar to those elicited by undiluted vaccine. CONCLUSIONS: Vaccination with VV elicits long-lived specific antibody responses directed against VV membrane proteins that vary by previous vaccination status but not with respect to 10-fold dilution of vaccine. B5, A33, and A27 should be considered for inclusion in future human orthopoxvirus subunit vaccines.

Reducing the dose of smallpox vaccine reduces vaccine-associated morbidity without reducing vaccination success rates or immune responses.

BACKGROUND: When the decision was made to prepare for a deliberate release of smallpox, the United States had approximately 15 million doses of Wyeth Dryvax vaccine, which was known to induce significant morbidity when used undiluted; Sanofi Pasteur, Inc., later identified approximately 85 million additional doses in storage. METHODS: Eleven vaccine-dose groups, each with 30 vaccinia-naive subjects, were given diluted Dryvax vaccine or 1 of 2 lots of Sanofi Pasteur smallpox vaccine and were evaluated for vaccination success rates, morbidity, and immune responses. RESULTS: Estimated doses of 10(6.6)-10(8.2) pfu of virus/mL induced major reactions (or "takes") in 93%-100% of subjects in each dose group. No differences in vaccination take rates, lesion size, erythema, and induration or in serum neutralizing-antibody response were detected between the groups. However, systemic reactogenicity and missed activities were significantly lower for the vaccine groups given doses of 10(6.6)-10(7.2) pfu/mL than for those given doses of 10(7.6)-10(8.2) pfu/mL. CONCLUSIONS: These findings support the use of a higher dilution of Wyeth Dryvax vaccine and Sanofi Pasteur smallpox vaccine, given that the resulting morbidity should be significantly lower without loss of vaccine effectiveness. A plan for use of higher dilutions would create an enormous stockpile of vaccine.

Flow-cytometric detection of vaccinia-induced memory effector CD4(+), CD8(+), and gamma delta TCR(+) T cells capable of antigen-specific expansion and effector functions.

We developed a carboxyfluorescein succinimidyl ester (CFSE)-based flow-cytometric assay that can detect different subsets of vaccinia-specific T cells capable of both antigen-specific expansion and protective effector functions. Proliferation and effector functions were detected by CFSE dilution and intracellular staining, respectively. Absolute numbers of CD4(+)/CFSE(lo)/interferon (IFN)- gamma (+), CD8(+)/CFSE(lo)/IFN- gamma (+), CD8(+)/CFSE(lo)/granzyme A(+), and CD8(+)/CFSE(lo)/CD107a(+) T cells present after in vitro stimulation with live vaccinia were significantly higher in immunized individuals (P<.05). These CD4(+) and CD8(+) T cell increases were >2 log higher than increases detectable by standard lymphoproliferation and cytotoxicity assays. Vaccinia-specific CD8(+)/CFSE(lo)/IFN- gamma (+) and granzyme A(+) T cell responses were significantly correlated with the results of standard (51)Cr-release cytolytic assays (P<.05). Furthermore, vaccinia induced antigen-specific memory gamma delta T cells. We demonstrate that vaccinia induces robust memory effector CD4(+), CD8(+), and gamma delta T cells, all of which are relevant for protection against smallpox. CFSE-based flow-cytometric assays will be useful in evaluating cell-mediated immune responses induced by new smallpox vaccines.

Stability of undiluted and diluted vaccinia-virus vaccine, Dryvax.

The stability of Dryvax vaccine was examined to determine whether diluted vaccine could be used over an extended period, thereby increasing the number of available doses. Stability parameters that we evaluated include dilution (undiluted, diluted 1:5, and diluted 1:10), diluent (original Dryvax diluent, new Dryvax diluent, and phosphate-buffered saline), and temperature (refrigerator temperature and room temperature). Storage of reconstituted Dryvax vaccine at room temperature, regardless of dilution or diluent, resulted in a significantly greater decrease in titer than did storage at refrigerator temperature. At refrigerator temperature, the decrease in vaccinia-vaccine titer during the first year was not significantly different between the undiluted Dryvax vaccine and either of the 2 diluted forms of Dryvax vaccine, with any of the 3 diluents.

Improved assay to detect neutralizing antibody following vaccination with diluted or undiluted vaccinia (Dryvax) vaccine.

The assessment of immunogenicity of a diluted vaccinia vaccine for possible widespread use of a diluted vaccine in the event of a bioterrorist attack prompted us to focus on the development of a sensitive and specific plaque reduction neutralization (PRN) assay to assess the antibody response of volunteers to a vaccinia (Dryvax) vaccine. Two incubation times, 1 h or overnight (approximately 15 h), were explored for the neutralization step of the assay. In addition, serum samples were evaluated using both sonicated and nonsonicated virus in PRN assays with 1 and 15 h of incubation. The use of the overnight incubation method resulted in the detection of antibody in two vaccinated individuals who exhibited a take, i.e., a major reaction indicative of successive vaccination as defined by the Centers for Disease Control and Prevention, but did not have a fourfold increase in antibody to vaccinia virus by the 1-h-incubation method and increased the sensitivity from 94 to 100%. In addition to the increased sensitivity of the assay, we noted a significant increase (approximately 40-fold) in the PRN titer of serum samples tested with the 15-h-incubation method. The use of sonicated virus increased the reproducibility of the virus titers and PRN titers. Forty-two percent of the samples tested using sonicated virus had a PRN titer that was fourfold higher or greater than that of nonsonicated virus in the assay. A PRN titer that was threefold higher or greater was observed in more than half (58%) of the samples using sonicated virus. Therefore, the more sensitive, specific, and reproducible plaque neutralization assay for the detection of antibody to vaccinia virus is the method using a 15-h-incubation time and freshly sonicated vaccinia virus.

Dose-dependent neutralizing-antibody responses to vaccinia.

To evaluate the humoral immune responses to smallpox-vaccine stocks currently available in the United States (Dryvax; Wyeth) and to generate data for comparison of responses to newly produced lots of smallpox vaccine, we evaluated dose-response effects, using undiluted and diluted smallpox vaccine. At 28 and 56 days after vaccination, serum samples were obtained from vaccinated subjects (N=674) who had participated in a randomized, single-blinded trial of an undiluted or a 1 : 5 or 1 : 10 dilution of smallpox vaccine and who subsequently were tested for plaque-reduction neutralizing-antibody titer. All subjects who developed a vesicle after vaccination also developed neutralizing antibodies by day 28. Subjects given either a 1 : 5 or 1 : 10 dilution of vaccinia had significantly higher neutralizing-antibody titers than did subjects given undiluted vaccine. Larger lesion size and fever after vaccination were associated with significantly higher neutralizing-antibody titers after vaccination.