Safety and immunogenicity of LC16m8, an attenuated smallpox vaccine in vaccinia-naive adults.

INTRODUCTION: LC16m8 is an attenuated cell culture-adapted Lister vaccinia smallpox vaccine missing the B5R protein and licensed for use in Japan. METHODS: We conducted a phase I/II clinical trial that compared the safety and immunogenicity of LC16m8 with Dryvax in vaccinia-naive participants. Adverse events were assessed, as were electrocardiography and laboratory testing for cardiotoxicity and viral culturing of the vaccination sites. Neutralization titers to vaccinia, monkeypox, and variola major were assessed and cell-mediated immune responses were measured by interferon (IFN)-γ enzyme-linked immunosorbent spot and lymphoproliferation assays. RESULTS: Local and systemic reactions after vaccination with LC16m8 were similar to those reported after Dryvax. No clinically significant abnormalities consistent with cardiac toxicity were seen for either vaccine. Both vaccines achieved antivaccinia, antivariola, and antimonkeypox neutralizing antibody titers >1:40, although the mean plaque reduction neutralization titer of LC16m8 at day 30 after vaccination was significantly lower than Dryvax for anti-NYCBH vaccinia (P < .01), antimonkeypox (P < .001), and antivariola (P < .001). LC16m8 produced robust cellular immune responses that trended higher than Dryvax for lymphoproliferation (P = .06), but lower for IFN-γ ELISPOT (P = .02). CONCLUSIONS: LC16m8 generates neutralizing antibody titers to multiple poxviruses, including vaccinia, monkeypox, and variola major, and broad T-cell responses, indicating that LC16m8 may have efficacy in protecting individuals from smallpox. Clinical Trials Registration. NCT00103584.

Clonal vaccinia virus grown in cell culture as a new smallpox vaccine.

Although the smallpox virus was eradicated over 20 years ago, its potential release through bioterrorism has generated renewed interest in vaccination. To develop a modern smallpox vaccine, we have adapted vaccinia virus that was derived from the existing Dryvax vaccine for growth in a human diploid cell line. We characterized six cloned and one uncloned vaccine candidates. One clone, designated ACAM1000, was chosen for development based on its comparability to Dryvax when tested in mice, rabbits and monkeys for virulence and immunogenicity. By most measures, ACAM1000 was less virulent than Dryvax. We compared ACAM1000 and Dryvax in a randomized, double-blind human clinical study. The vaccines were equivalent in their ability to produce major cutaneous reactions ('takes') and to induce neutralizing antibody and cell-mediated immunity against vaccinia virus.

Quantitation of CD8+ T cell responses to newly identified HLA-A*0201-restricted T cell epitopes conserved among vaccinia and variola (smallpox) viruses.

Immunization with vaccinia virus resulted in long-lasting protection against smallpox and was the approach used to eliminate natural smallpox infections worldwide. Due to the concern about the potential use of smallpox virus as a bioweapon, smallpox vaccination is currently being reintroduced. Severe complications from vaccination were associated with congenital or acquired T cell deficiencies, but not with congenital agammaglobulinemia, suggesting the importance of T cell immunity in recovery from infection. In this report, we identified two CD8+ T cell epitopes restricted by the most common human major histocompatibility complex (MHC) class I allele, HLA-A*0201. Both epitopes are highly conserved in vaccinia and variola viruses. The frequency of vaccinia-specific CD8+ T cell responses to these epitopes measured by interferon (IFN)-gamma enzyme-linked immunospot (ELISPOT) assay and HLA/peptide tetramer staining peaked 2 wk after primary immunization and then declined, but were still detectable 1 to 3 yr after primary immunization. 2 wk after immunization, IFN-gamma-producing cells specific to these two epitopes were 14% of total vaccinia virus-specific IFN-gamma-producing cells in one donor, 35% in the second donor, and 6% in the third donor. This information will be useful for studies of human T cell memory and for the design and analyses of the immunogenicity of experimental vaccinia vaccines.

Multifunctional T-cell characteristics induced by a polyvalent DNA prime/protein boost human immunodeficiency virus type 1 vaccine regimen given to healthy adults are dependent on the route and dose of administration.

A phase I clinical vaccine study of a human immunodeficiency virus type 1 (HIV-1) vaccine regimen comprising a DNA prime formulation (5-valent env and monovalent gag) followed by a 5-valent Env protein boost for seronegative adults was previously shown to induce HIV-1-specific T cells and anti-Env antibodies capable of neutralizing cross-clade viral isolates. In light of these initial findings, we sought to more fully characterize the HIV-1-specific T cells by using polychromatic flow cytometry. Three groups of participants were vaccinated three times with 1.2 mg of DNA administered intradermally (i.d.; group A), 1.2 mg of DNA administered intramuscularly (i.m.; group B), or 7.2 mg of DNA administered i.m. (high-dose group C) each time. Each group subsequently received one or two doses of 0.375 mg each of the gp120 protein boost vaccine (i.m.). Env-specific CD4 T-cell responses were seen in the majority of participants; however, the kinetics of responses differed depending on the route of DNA administration. The high i.m. dose induced the responses of the greatest magnitude after the DNA vaccinations, while the i.d. group exhibited the responses of the least magnitude. Nevertheless, after the second protein boost, the magnitude of CD4 T-cell responses in the i.d. group was indistinguishable from those in the other two groups. After the DNA vaccinations and the first protein boost, a greater number of polyfunctional Env-specific CD4 T cells (those with > or = 2 functions) were seen in the high-dose group than in the other groups. Gag-specific CD4 T cells and Env-specific CD8 T cells were seen only in the high-dose group. These findings demonstrate that the route and dose of DNA vaccines significantly impact the quality of immune responses, yielding important information for future vaccine design.

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.

Safety and immunogenicity of new cell-cultured smallpox vaccine compared with calf-lymph derived vaccine: a blind, single-centre, randomised controlled trial.

BACKGROUND: US government organisations have identified the need for a new smallpox vaccine to replenish limited stocks of the approved, calf-lymph derived vaccine, the manufacture of which is no longer acceptable. We aimed to compare the safety and immunogenicity of the new cell-cultured smallpox vaccine (CCSV) to that of the calf-lymph derived vaccine (as a positive control) in 350 healthy, adult volunteers. METHODS: We did a randomised controlled study at the University of Kentucky Medical Center. We randomised 150 vaccinia-naive volunteers, aged 18-30 years, and 100 vaccinia-non-naive people, aged 32-65 years, to equivalent doses of either CCSV or test vaccine (2.5x10(5) plaque-forming units) by 15 puncture scarification in double-blind fashion. Immunogenicity was assessed by pock formation (take rate), humoral immune response by plaque-reduction neutralisation titres, and cellular immune response by vaccinia-specific, interferon-gamma T-cell quantification, cytotoxicity, and T-cell proliferation response. A further 100 vaccine-naive individuals, aged 18-30 years, received one of five doses of CCSV (undiluted, diluted 1 in 5, 1 in 10, 1 in 25, and 1 in 50) in single-blind fashion. Routine laboratory assessments, physical examinations, and recording of adverse events were done to assess vaccine safety. The primary endpoints were safety and reactogenicity (take rate) of CCSV. FINDINGS: 349 (99.7%) of 350 volunteers developed pock lesions; one vaccinia-naive individual who received a 1 in 25 dilution of CCSV did not. The rate of adverse events related to vaccine and the extent of humoral and cellular immune responses did not differ between the vaccine groups in vaccinia-naive or non-naive people. CCSV was immunogenic in vaccine-naive volunteers at a dose 50 times lower than that approved for Dryvax. INTERPRETATION: CCSV seems to be a safe and immunogenic alternative to calf-lymph derived vaccine for both vaccinia-naive and non-naive people.

Identification of vaccinia CD8+ T-cell epitopes conserved among vaccinia and variola viruses restricted by common MHC class I molecules, HLA-A2 or HLA-B7.

Immunization with vaccinia virus results in long-lasting protection against smallpox and is an approach that has been successfully used to eliminate natural smallpox infections worldwide. Today, vaccinia virus is very important not only as a vaccine virus to protect human against smallpox, but also as an expression vector for immunization against other infectious diseases, such as HIV and cancer. In this article, we identify three new vaccinia human CD8+ T-cell epitopes conserved among vaccinia and variola viruses restricted by HLA-A2, HLA-B7, or HLA-B*3502, which belongs to the HLA-B7 supertype. Identification of these CD8+ T-cell epitopes restricted by common HLA alleles will help to quantitate human CD8+ T-cell responses to licensed and experimental smallpox vaccines and to vaccinia virus vectors. CD8+ T-cell responses specific to these epitopes can also be used to quantitate cellular immune responses, especially with new smallpox vaccines that do not induce a "take," such as the modified vaccinia virus Ankara strain. Combined with previous reports by us and others, these results show that there are some vaccinia viral proteins containing multiple epitopes restricted by different MHC molecules of humans and mice.

Expression and function of TLR2, TLR4, and Nod2 in primary canine colonic epithelial cells.

The gut maintains a delicate balance between the downregulation of inflammatory reactions to commensal bacteria and the capacity to respond to pathogens with vigorous cellular and humoral immune responses. Intestinal epithelial cells, including colonic epithelial cells (CECs) possess many properties of cells of the innate immune system, in particular the ability to recognize and respond to microbial antigens. Recognition of microorganisms by CECs is based upon their recognition of signature molecules, called microbe-associated molecular patterns (MAMP), by pattern recognition receptors (PRR) including membrane toll-like receptors (TLR) and cytosolic Nod2, an intracellular counterpart of TLRs. The purpose of this study was to determine whether primary CECs from normal dogs express a functional TLR2, TLR4, and Nod2 and whether they are regulated by inflammatory mediators. We show that canine primary CECs express TLR2, TLR4, and Nod2 that can be modulated in response to their respective MAMPs, lipopolysaccharides (LPS) or peptidoglycans (PGN). Furthermore, we demonstrate that these receptors are functional as evidenced by the induction of cytokine gene expression in response to LPS or PGN.

Role of T-regulatory cells in the response to hepatitis B vaccine in hemodialysis patients.

Human disease elicits a complex array of biological processes that results in long-term protective immunological memory to infectious agents. Chronic kidney disease is known to impair induction of sustained immunological memory to hepatitis B vaccine (HBVax) antigens. We asked the question: Does end-stage renal disease promote changes in subtypes of regulatory T (Treg) cells that correlate with diminished amnestic response to HBVax antigen compared to healthy controls? The study design and setting was a prospective observational cohort at a veterans affairs medical center. End-stage renal disease patients on hemodialysis (HD) were compared with individuals with self-reported normal kidney function. All subjects received HBVax. Peripheral blood was sampled for assessment for Treg cells pre and post vaccination. CD4+ FOXP3 Treg numbers were similar between HD and healthy subjects during a 14-day time period post vaccination. HD subjcts had lower anti-HBSag antibody than CON (control) subjects (330 ± 108.7 vs. 663.1 ± 129.7 IU/mL; P = 0.063). Hemodialysis subjects with resting Tregs higher than the median value in our cohort demonstrated a significantly lower change in HBsAB at 30 days post booster vaccination (P = 0.030). No such relationship was found for the activated Treg subset among HD subjects, or either subset among CON subsets. In our limited comparison study of 11 HD and 8 CON subjects, Treg subsets did not differ between the two groups; but differences in the suppressive Treg numbers in the HD group could explain the altered antibody response to HBVax and is worthy of further study.

Novel nutritional immune formula maintains host defense mechanisms.

Military combat and training stress induce immune changes that increase the risk of infection and ultimately influence soldiers' performance and readiness. Strenuous military training/assessment provides a uniform stress and the opportunity to evaluate nutritional strategies to minimize stress-induced immune changes that predispose soldiers to infection. Immunological changes and effects of a novel nutritional immune formula (NNIF) were examined prospectively in a double-blind, controlled study of 200 soldiers attending Special Forces Assessment and Selection School. Immune function was measured by skin delayed-type hypersensitivity, lymphocyte phenotyping, mitogenic proliferative responses, and granulocyte function. Approximately 50% of soldiers completed the study (control, n = 57; NNIF, n = 50). Several stress-induced lymphocyte changes were observed (decreased mitogen-induced proliferation, T and total lymphocytes, and interferon-gamma-producing lymphocytes and increased percentage of neutrophils). NNIF modified several changes, including delayed-type hypersensitivity responses (NNIF, 78%; control, 59%; p < 0.05), increased proportions of helper T cells, activation of B cells, enhanced neutrophil phagocytosis, and attenuation of declines in certain functional subpopulations (i.e., cytotoxic/ suppressor lymphocytes). Soldiers who consumed NNIF experienced less stress-induced immune impairment, thereby lowering the risk of infection.

Vaccination issues in patients with chronic kidney disease.

Infections are an important cause of morbidity and mortality among patients at all stages of chronic kidney disease. Prevention through vaccination remains the best strategy to minimize the adverse consequences associated with these infectious diseases in this, and all, populations. Unfortunately, patients with chronic kidney disease demonstrate inadequacies of specific immune-cell function that are required for generating a protective vaccine response. Nevertheless, early vaccination of this high-risk population has demonstrated good clinical outcomes during progression to late-stage disease. We review the available evidence linking immune impairment in adult patients with late-stage chronic kidney disease to diminished vaccine responses. We highlight the importance of early vaccination in disease with high risk for development of CKD and novel vaccine approaches in development that may help to address improvement in protective boosting of immunity during late-stage disease.

Diverting T helper cell trafficking through increased plasticity attenuates autoimmune encephalomyelitis.

Naive T helper cells differentiate into functionally distinct effector subsets that drive specialized immune responses. Recent studies indicate that some of the effector subsets have plasticity. Here, we used an EAE model and found that Th17 cells deficient in the transcription factor BCL11B upregulated the Th2-associated proteins GATA3 and IL-4 without decreasing RAR-related orphan receptor γ (RORγt), IL-17, and GM-CSF levels. Surprisingly, abnormal IL-4 production affected Th17 cell trafficking, diverting migration from the draining lymph nodes/CNS route to the mesenteric lymph nodes/gut route, which ameliorated EAE without overt colitis. T helper cell rerouting in EAE was dependent on IL-4, which enhanced retinoic acid (RA) production by dendritic cells, which further induced expression of gut-homing receptors CCR9 and α4ß7 on Bcl11b-deficient CD4+ T cells. Furthermore, IL-4 treatment or Th2 immunization of wild-type mice with EAE caused no alteration in Th17 cytokines or RORγt, but diverted T helper cell trafficking to the gut, which improved EAE outcome without overt colitis. Our data demonstrate that Th17 cells are permissive to Th2 gene expression without affecting Th17 gene expression. This Th17 plasticity has an impact on trafficking, which is a critical component of the immune response and may represent a possible avenue for treating multiple sclerosis.

Evaluation of potential genotoxicity of HIV entry inhibitors derived from natural sources.

AIDS is a global pandemic that has seen the development of novel and effective treatments to improve the quality of life of those infected and reduction of spread of the disease. Palmitic Acid (PA), which we identified and isolated from Sargassum fusiforme, is a naturally occurring fatty acid that specifically inhibits HIV entry by binding to a novel pocket on the CD4 receptor. We also identified a structural analogue, 2-bromopalmitate (2-BP), as a more effective HIV entry inhibitor with a 20-fold increase in efficacy. We have used the structure-activity relationship (SAR) of 2-BP as a platform to identify new small chemical molecules that fit into the various identified active sites in an effort to identify more potent CD4 entry inhibitors. To validate further drug development, we tested the PA and 2-BP scaffold molecules for genotoxic potential. The FDA and International Conference on Harmonisation (ICH) recommends using a standardized 3-test battery for testing compound genotoxicity consisting of the bacterial reverse mutation assay, mouse lymphoma assay, and rat micronucleus assay. PA and 2-BP and their metabolites tested negative in all three genotoxicty tests. 2-BP is the first derivative of PA to undergo pre-clinical screening, which will enable us to now test multiple simultaneous small chemical structures based on activity in scaffold modeling across the dimension of pre-clinical testing to enable transition to human testing.

Ex vivo γ-retroviral gene therapy of dogs with X-linked severe combined immunodeficiency and the development of a thymic T cell lymphoma.

We have previously shown that in vivo γ-retroviral gene therapy of dogs with X-linked severe combined immunodeficiency (XSCID) results in sustained T cell reconstitution and sustained marking in myeloid and B cells for up to 4 years with no evidence of any serious adverse effects. The purpose of this study was to determine whether ex vivo γ-retroviral gene therapy of XSCID dogs results in a similar outcome. Eight of 12 XSCID dogs treated with an average of dose of 5.8 × 10(6) transduced CD34(+) cells/kg successfully engrafted producing normal numbers of gene-corrected CD45RA(+) (naïve) T cells. However, this was followed by a steady decrease in CD45RA(+) T cells, T cell diversity, and thymic output as measured by T cell receptor excision circles (TRECs) resulting in a T cell lymphopenia. None of the dogs survived past 11 months post treatment. At necropsy, few gene-corrected thymocytes were observed correlating with the TREC levels and one of the dogs was diagnosed with a thymic T cell lymphoma that was attributed to the gene therapy. This study highlights the outcome differences between the ex vivo and in vivo approach to γ-retroviral gene therapy and is the first to document a serious adverse event following gene therapy in a canine model of a human genetic disease.

IMVAMUNE: modified vaccinia Ankara strain as an attenuated smallpox vaccine.

Smallpox vaccines based on replicating vaccinia virus are known to elicit rare yet serious adverse events, particularly in human populations with immune deficiency, atopic dermatitis and at the extremes of age. A vaccine that induces protective immune responses equivalent to first-generation smallpox vaccines while reducing the risk for severe adverse events is critical for a national stockpile of smallpox vaccines. Modified vaccinia Ankara (MVA) has been proposed as an immediate solution for vaccination of high-risk individuals. Bavarian Nordic's vaccine MVA-BN (IMVAMUNE) is a MVA strain that is replication incompetent in mammalian cell lines. IMVAMUNE has been administered to more than 1900 human subjects to date, including high-risk populations (e.g., people diagnosed with atopic dermatitis or infected with HIV) in which standard replicating vaccines are contraindicated. We review the Phase I clinical trial safety profile and immune responses and compare them with other smallpox vaccines, including ACAM2000 and Dryvax.

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.

ACAM2000: a newly licensed cell culture-based live vaccinia smallpox vaccine.

BACKGROUND: Due to concern over i) expiration of currently available calf-lymph vaccine (Dryvax); ii) calf lymph as a vaccine (bovine spongiform encephalopathy [BSE], other possible contaminations and animal welfare); and iii) use of variola as a weapon for bioterrorism, a new and safer vaccinia-based smallpox vaccine derived from new cell culture-based technology was proposed. Federally funded work by Acambis, Inc. resulted in FDA approval for ACAM2000 in August 2007. OBJECTIVES: This paper describes the development from conception to FDA approval of the new vaccinia cell cultured-based smallpox vaccine ACAM2000. METHODS: Data were compiled from available public reports. RESULTS/CONCLUSIONS: The studies with ACAM2000 indicate that it closely matches the safety of Dryvax in both non-clinical and clinical trials. ACAM2000 met two of the four primary surrogate efficacy end point criteria established for the Phase III clinical trials. Concern over the incidence of myopericarditis with ACAM2000 and Dryvax exists. So far the cardiac events seem to be self-limited. There are no pediatric safety data for ACAM2000. Overall, clinical trial results were sufficient to convince the FDA that ACAM2000 is a suitable replacement for Dryvax in the event of bioterrorism involving variola (smallpox).

Cross-subtype antibody and cellular immune responses induced by a polyvalent DNA prime-protein boost HIV-1 vaccine in healthy human volunteers.

An optimally effective AIDS vaccine would likely require the induction of both neutralizing antibody and cell-mediated immune responses, which has proven difficult to obtain in previous clinical trials. Here we report on the induction of Human Immunodeficiency Virus Type-1 (HIV-1)-specific immune responses in healthy adult volunteers that received the multi-gene, polyvalent, DNA prime-protein boost HIV-1 vaccine formulation, DP6-001, in a Phase I clinical trial conducted in healthy adult volunteers of both genders. Robust cross-subtype HIV-1-specific T cell responses were detected in IFNgamma ELISPOT assays. Furthermore, we detected high titer serum antibody responses that recognized a wide range of primary HIV-1 Env antigens and also neutralized pseudotyped viruses that express the primary Env antigens from multiple HIV-1 subtypes. These findings demonstrate that the DNA prime-protein boost approach is an effective immunization method to elicit both humoral and cell-mediated immune responses in humans, and that a polyvalent Env formulation could generate broad immune responses against HIV-1 viruses with diverse genetic backgrounds.

Cross-subtype antibody and cellular immune responses induced by a polyvalent DNA prime-protein boost HIV-1 vaccine in healthy human volunteers.

An optimally effective AIDS vaccine would likely require the induction of both neutralizing antibody and cell-mediated immune responses, which has proven difficult to obtain in previous clinical trials. Here we report on the induction of human immunodeficiency virus type-1 (HIV-1)-specific immune responses in healthy adult volunteers that received the multi-gene, polyvalent, DNA prime-protein boost HIV-1 vaccine formulation, DP6-001, in a Phase I clinical trial. Robust cross-subtype HIV-1 specific T cell responses were detected in IFN-gamma ELISPOT assays. Furthermore, we detected high titer serum antibody responses that recognized a wide range of primary HIV-1 Env antigens and also neutralized pseudotyped viruses that express the primary Env antigens from multiple HIV-1 subtypes. These findings demonstrate that the DNA prime-protein boost approach is an effective immunization method to elicit both humoral and cell-mediated immune responses in humans, and that a polyvalent Env formulation could generate broad immune responses against HIV-1 viruses with diverse genetic backgrounds.