Jeffrey G. Smith

Comparative Immunogenicity in Rhesus Monkeys of DNA Plasmid, Recombinant Vaccinia Virus, and Replication-Defective Adenovirus Vectors Expressing a Human Immunodeficiency Virus Type 1 gag Gene

ABSTRACT Cellular immune responses, particularly those associated with CD3+ CD8+ cytotoxic T lymphocytes (CTL), play a primary role in controlling viral infection, including persistent infection with human immunodeficiency virus type 1 (HIV-1). Accordingly, recent HIV-1 vaccine research efforts have focused on establishing the optimal means of eliciting such antiviral CTL immune responses. We evaluated several DNA vaccine formulations, a modified vaccinia virus Ankara vector, and a replication-defective adenovirus serotype 5 (Ad5) vector, each expressing the same codon-optimized HIV-1 gag gene for immunogenicity in rhesus monkeys. The DNA vaccines were formulated with and without one of two chemical adjuvants (aluminum phosphate and CRL1005). The Ad5-gag vector was the most effective in eliciting anti-Gag CTL. The vaccine produced both CD4+ and CD8+ T-cell responses, with the latter consistently being the dominant component. To determine the effect of existing antiadenovirus immunity on Ad5-gag-induced immune responses, monkeys were exposed to adenovirus subtype 5 that did not encode antigen prior to immunization with Ad5-gag. The resulting anti-Gag T-cell responses were attenuated but not abolished. Regimens that involved priming with different DNA vaccine formulations followed by boosting with the adenovirus vector were also compared. Of the formulations tested, the DNA-CRL1005 vaccine primed T-cell responses most effectively and provided the best overall immune responses after boosting with Ad5-gag. These results are suggestive of an immunization strategy for humans that are centered on use of the adenovirus vector and in which existing adenovirus immunity may be overcome by combined immunization with adjuvanted DNA and adenovirus vector boosting.

Varicella-Zoster Virus–Specific Immune Responses in Elderly Recipients of a Herpes Zoster Vaccine

BACKGROUND A double-blind, placebo-controlled trial that involved 38,546 subjects > or =60 years old demonstrated efficacy of a high-potency live-attenuated Oka/Merck varicella-zoster virus (VZV) vaccine. The trial included an immunology substudy to determine the relationship of VZV-specific immune responses to vaccination and clinical outcome. METHODS The immunology substudy enrolled 1395 subjects at 2 sites where blood samples obtained prior to vaccination, at 6 weeks after vaccination, and at 1, 2, and 3 years thereafter were tested for VZV-specific cell-mediated immunity (VZV-CMI) by gamma-interferon ELISPOT and responder cell frequency assays and for VZV antibody by glycoprotein ELISA. RESULTS VZV-CMI and VZV antibodies were significantly increased in vaccine recipients at 6 weeks after vaccination. The vaccine-induced increases in VZV-CMI persisted during the 3 years of follow-up, although their magnitude decreased over time. The magnitude of these VZV-specific immune responses was greater in subjects 60-69 years old than in subjects > or =70 years old. CONCLUSIONS The zoster vaccine induced a significant increase in VZV-CMI and VZV antibody. The magnitude and duration of the boost in VZV-CMI in vaccine recipients and the relationship of this boost to age paralleled the clinical effects of the vaccine observed during the efficacy trial. These findings support the hypothesis that boosting VZV-CMI protects older adults against herpes zoster and postherpetic neuralgia.

Decline in Varicella-Zoster Virus (VZV)–Specific Cell-Mediated Immunity with Increasing Age and Boosting with a High-Dose VZV Vaccine

The safety and immunogenecity of a booster dose of live attenuated varicella-zoster virus (VZV) vaccine was evaluated in 196 healthy subjects, >or=60 years old, who had already received a VZV vaccine >5 years before. This repeat booster dose was well tolerated. Cell-mediated immunity (CMI) to VZV was measured by an interferon-gamma (IFN-gamma) enzyme-linked immunosorbent spot-forming cell (ELISPOT) assay and a limiting dilution responder cell frequency (RCF) assay. Prevaccination responses decreased as a function of increasing age but were detectable in all subjects by use of the IFN-gamma ELISPOT assay. In most subjects, VZV-specific CMI was increased at 6 weeks postvaccination. The magnitude of the vaccine-induced IFN-gamma ELISPOT response was inversely related to prevaccination values. Although there was a significant correlation between the IFN-gamma ELISPOT and RCF assays, the ELISPOT assay had greater sensitivity and a wider dynamic range. A live attenuated VZV vaccine is safe and immunogenic in an elderly population, and the vaccine-induced immunity may be monitored by the IFN-gamma ELISPOT assay.

Varicella-Zoster Virus–Specific Immune Responses to Herpes Zoster in Elderly Participants in a Trial of a Clinically Effective Zoster Vaccine

BACKGROUND The objectives of this study were to evaluate the association between varicella-zoster virus (VZV)-specific humoral and cell-mediated immunity (CMI) to herpes zoster (HZ) and protection against HZ morbidity and to compare immune responses to HZ and zoster vaccine. METHODS In 981 elderly persons who developed HZ during a zoster vaccine efficacy trial (321 vaccinees and 660 placebo recipients) and 1362 without HZ (682 vaccinees and 680 placebo recipients), CMI was measured by VZV responder cell frequency and interferon-gamma enzyme-linked immunospot, and antibodies were measured by VZV enzyme-linked immunosorbent assay against affinity-purified VZV glycoproteins (gpELISA). RESULTS Robust VZV CMI at HZ onset correlated with reduced HZ morbidity, whereas VZV gpELISA titers did not. Three weeks after HZ onset, gpELISA titers were highest in those with more severe HZ and were slightly increased in placebo recipients (compared with zoster vaccine recipients) and in older individuals. VZV CMI responses to HZ were similar in zoster vaccine and placebo recipients and were not affected by demographic characteristics or antiviral therapy, except for responder cell frequency at HZ onset, which decreased with age. When responses to zoster vaccine and HZ could be compared, VZV CMI values were similar, but antibody titers were lower. CONCLUSIONS Higher VZV CMI at HZ onset was associated with reduced HZ severity and less postherpetic neuralgia. Higher antibody titers were associated with increased HZ severity and occurrence of postherpetic neuralgia. HZ and zoster vaccine generated comparable VZV CMI.

Development and Validation of a Gamma Interferon ELISPOT Assay for Quantitation of Cellular Immune Responses to Varicella-Zoster Virus

ABSTRACT Cell-mediated immunity appears to be critical for the prevention and control of varicella-zoster virus (VZV) infection and complications arising from zoster. Current assays of VZV-specific cell-mediated immunity are cumbersome or lack sensitivity. We have developed a gamma interferon ELISPOT assay that provides a direct measure of the number of T cells secreting a cytokine following stimulation with antigen. This assay is extremely sensitive and specific, with the ability to detect gamma interferon spot-forming cells (SFC) in the range of 10 to 1,000 SFC per million peripheral blood mononuclear cells (PBMCs). This assay has been validated by demonstrating the following: (i) the response detected is mediated almost entirely by CD4+ T cells, (ii) ELISPOT responses from fresh-frozen PBMCs are equivalent to those from freshly isolated cells, (iii) frozen PBMCs can be shipped on dry ice for up to 48 h without loss of activity, (iv) frozen PBMC samples can be stored in liquid nitrogen over long periods (>22 months) without any significant change in response, and (v) the numbers of ELISPOTs counted using a computer-based imaging system are equivalent to those counted by humans but have lower variability. The ability to use frozen cells is facilitated by the use of a recombinant nuclease (Benzonase) that can prevent cell clumping when samples are thawed. Frozen PBMC samples can be cycled through multiple changes in storage between liquid nitrogen and dry ice without any change in response being detected. This facilitates collection of samples at one site and testing performed at a remote location. This VZV ELISPOT assay provides a new versatile tool for monitoring cellular immune responses either during a herpes zoster disease outbreak or following vaccination.

Efficacy of the Echinocandin Caspofungin against Disseminated Aspergillosis and Candidiasis in Cyclophosphamide-Induced Immunosuppressed Mice

ABSTRACT The in vivo efficacy of the echinocandin antifungal caspofungin acetate (caspofungin; MK-0991) was evaluated in models of disseminated aspergillosis and candidiasis in mice with cyclophosphamide (CY)-induced immunosuppression. Caspofungin is a 1,3-β-d-glucan synthesis inhibitor efficacious against a number of clinically relevant fungi including Aspergillusand Candida species. Models of CY-induced transient or chronic leukopenia were used with once daily administration of therapy initiated 24 h after microbial challenge. Caspofungin was effective in treating disseminated aspergillosis in mice that were transiently leukopenic (significant prolongation of survival at doses of ≥0.125 mg/kg of body weight and a 50% protective dose [PD50] of 0.245 mg/kg/day at 28 days after challenge) or chronically leukopenic (50 to 100% survival at doses of ≥0.5 mg/kg and PD50s ranging from 0.173 to 0.400 mg/kg/day). Caspofungin was effective in the treatment and sterilization ofCandida infections in mice with transient leukopenia with a 99% effective dose based on reduction in log10 CFU ofCandida albicans/gram of kidneys of 0.119 mg/kg and 80 to 100% of the caspofungin-treated mice having sterile kidneys at caspofungin doses from 0.25 to 2.0 mg/kg. InCandida-infected mice with chronic leukopenia, caspofungin was effective at all dose levels tested (0.25 to 1.0 mg/kg), with the log10 CFU of C. albicans/gram of kidneys of caspofungin-treated mice being significantly lower (>99% reduction) than that of sham-treated mice from day 4 to day 28 after challenge. Also, 70 to 100% of the caspofungin-treated, chronic leukopenic mice had sterile kidneys at caspofungin doses of 0.5 to 1.0 mg/kg from day 8 to 28 after challenge. Sterilization of Candida infections by caspofungin in the absence of host leukocytes provides compelling in vivo evidence for fungicidal activity against C. albicans. Further human clinical trials with caspofungin against serious fungal infections are in progress.

Evaluation of water-soluble pneumocandin analogs L-733560, L-705589, and L-731373 with mouse models of disseminated aspergillosis, candidiasis, and cryptococcosis.

The activities of the water-soluble pneumocandin derivatives L-733560, L-705589, and L-731373 were evaluated in mouse models of disseminated aspergillosis, candidiasis, and cryptococcosis and were compared with those of commercially available antifungal agents. Pneumocandins are inhibitors of 1,3-beta-D-glucan synthesis. In the aspergillosis model, L-733560 and L-705589 significantly prolonged the survival of DBA/2N mice challenged intravenously with Aspergillus fumigatus conidia. L-733560 and L-705589 exhibited efficacies comparable to that of amphotericin B (AMB) with 90% effective doses of 0.48, 0.12, and 0.36 mg/kg of body weight, respectively. Two mouse models of disseminated candidiasis were used to evaluate these compounds. In both models, mice were challenged intravenously with Candida albicans. In a C. albicans survival model with DBA/2N and CD-1 mice, the efficacy of L-733560 was comparable to that of AMB, while L-731373 and L-705589 were somewhat less active. In a previously described C. albicans target organ kidney assay, the pneumocandin analogs and AMB at doses of > or = 0.09 mg/kg were effective in sterilizing kidneys, while fluconazole and ketoconazole were considerably less active and did not sterilize kidneys when they were used at concentrations of < or = 100 mg/kg. Although orally administered L-733560 showed activity in both candidiasis models, its efficacy was reduced compared with that of parenterally administered drug. In a disseminated cryptococcosis mouse model that measures the number of CFU of Cryptococcus neoformans per gram of brain and spleen, L-733560 at 10 mg/kg was ineffective in reducing the counts in organs, while AMB at 0.31 mg/kg sterilized the organs. These results indicate that the pneumocandins may be beneficial as potent parenterally administered therapeutic agents for disseminated aspergillosis and candidiasis.

Establishing Acceptance Criteria for Cell-Mediated-Immunity Assays Using Frozen Peripheral Blood Mononuclear Cells Stored under Optimal and Suboptimal Conditions

ABSTRACT The enzyme-linked immunospot (ELISPOT) assay is a powerful tool for measuring antigen-specific cellular immune responses. The ability to use frozen peripheral blood mononuclear cells (PBMC) facilitates testing samples in multicenter clinical trials; however, unreliable ELISPOT responses may result if samples are not handled properly. Exposure of frozen PBMC to suboptimal storage temperature (−20°C) or repeated cycling between more optimal storage temperatures (less than −130°C and −70°C) reduced the quality of frozen PBMC, as assessed by cell viability and functional ELISPOT response measures. Cell viability as assessed by trypan blue dye exclusion was reduced, and the percentage of apoptotic cells, as determined by the Guava Nexin assay, was significantly increased after these events. The functional gamma interferon ELISPOT responses to phytohemagglutinin (PHA) mitogen, a CD4 T-cell-specific antigen (varicella-zoster virus), and a CD8 T-cell-specific antigen (pool containing known cytomegalovirus, Epstein-Barr virus, and influenza virus peptides) were all significantly reduced after suboptimal storage events. However, for a given suboptimal storage event, the magnitude of the reduction varied between individuals and even among aliquots within an individual bleed, indicating the need for sample-specific acceptance criteria (AC). The percent viable or percent apoptotic cells after thaw, as well as the functional ELISPOT response to PHA, were all effective when applied with limits as AC for separating samples damaged during storage from valid control samples. Although all three AC measures could be effectively applied, the apoptosis AC limit applied was best for separating samples that could respond to antigenic stimulation from samples that could not effectively respond.

Safety, tolerability, and immunogenicity after 1 and 2 doses of zoster vaccine in healthy adults ≥60 years of age

BACKGROUND Incidence and severity of herpes zoster (HZ) and postherpetic neuralgia increase with age, associated with age-related decrease in immunity to varicella-zoster virus (VZV). One dose of zoster vaccine (ZV) has demonstrated substantial protection against HZ; this study examined impact of a second dose of ZV. METHODS Randomized, double-blind, multicenter study with 210 subjects ≥60 years old compared immunity and safety profiles after one and two doses of ZV, separated by 6 weeks, vs. placebo. Immunogenicity was evaluated using VZV interferon-gamma (IFN-γ) enzyme-linked immunospot (ELISPOT) assay and VZV glycoprotein enzyme-linked immunosorbent antibody (gpELISA) assay. Adverse experiences (AEs) were recorded on a standardized Vaccination Report Card. RESULTS No serious vaccine-related AEs occurred. VZV IFN-γ ELISPOT geometric mean count (GMC) of spot-forming cells per 10(6) peripheral blood mononuclear cells increased in the ZV group from 16.9 prevaccination to 49.5 and 32.8 at 2 and 6 weeks postdose 1, respectively. Two weeks, 6 weeks and 6 months postdose 2, GMC was 44.3, 42.9, and 36.5, respectively. GMC in the placebo group did not change during the study. The peak ELISPOT response occurred ∼2 weeks after each ZV dose. The gpELISA geometric mean titers (GMTs) in the ZV group were higher than in the placebo group at 6 weeks after each dose. Correlation between the IFN-γ ELISPOT and gpELISA assays was poor. CONCLUSIONS ZV was generally well-tolerated and immunogenic in adults ≥60 years old. A second dose of ZV was generally safe, but did not boost VZV-specific immunity beyond levels achieved postdose 1.

Major depressive disorder and immunity to varicella-zoster virus in the elderly.

Major depressive disorder has been associated with activation of inflammatory processes as well as with reductions in innate, adaptive and non-specific immune responses. The objective of this study was to evaluate the association between major depression and a disease-relevant immunologic response, namely varicella-zoster virus (VZV)-specific immunity, in elderly adults. A cross-sectional cohort study was conducted in 104 elderly community dwelling adults ≥ 60years of age who were enrolled in the depression substudy of the shingles prevention study, a double blind, placebo-controlled vaccine efficacy trial. Fifty-two subjects had a current major depressive disorder, and 52 age- and sex-matched controls had no history of depression or any mental illness. VZV-specific cell-mediated immunity (VZV-CMI) was measured by VZV responder cell frequency (VZV-RCF) and interferon-γ enzyme-linked immunospot (ELISPOT) assays, and antibody to VZV was measured by an enzyme-linked immunosorbent assay against affinity-purified VZV glycoproteins (gpELISA). VZV-CMI, measured by VZV-RCF, was significantly lower in the depressed group than in the controls (p<0.001), and VZV-RCF was inversely correlated with the severity of depressive symptoms in the depressed patients. In addition, an age-related reduction in VZV-RCF was observed in the depressed patients, but not in the controls. Furthermore, there was a trend for depressive symptom severity to be associated with lower ELISPOT counts. Finally, VZV-RCF was higher in depressed patients treated with antidepressant medications as compared to untreated depressed patients. Since lower levels of VZV-RCF appear to explain the increased risk and severity of herpes zoster observed in older adults, these findings suggest that, in addition to increasing age, depression may increase the risk and severity of herpes zoster.