Interferon-gamma Enzyme-linked Immunospot Assay Research Articles

Vaccine protection against simian immunodeficiency virus in monkeys using recombinant gamma-2 herpesvirus.

Recombinant strains of replication-competent rhesus monkey rhadinovirus (RRV) were constructed in which strong promoter/enhancer elements were used to drive expression of simian immunodeficiency virus (SIV) Env or Gag or a Rev-Tat-Nef fusion protein. Cultured rhesus monkey fibroblasts infected with each recombinant strain were shown to express the expected protein. Three RRV-negative and two RRV-positive rhesus monkeys were inoculated intravenously with a mixture of these three recombinant RRVs. Expression of SIV Gag was readily detected in lymph node biopsy specimens taken at 3 weeks postimmunization. Impressive anti-SIV cellular immune responses were elicited on the basis of major histocompatibility complex (MHC) tetramer staining and gamma interferon enzyme-linked immunospot (ELISPOT) assays. Responses were much greater in magnitude in the monkeys that were initially RRV negative but were still readily detected in the two monkeys that were naturally infected with RRV at the time of immunization. By 3 weeks postimmunization, responses measured by MHC tetramer staining in the two Mamu-A*01(+) RRV-negative monkeys reached 9.3% and 13.1% of all CD8(+) T cells in peripheral blood to the Gag CM9 epitope and 2.3% and 7.3% of all CD8(+) T cells in peripheral blood to the Tat SL8 epitope. Virus-specific CD8(+) T cell responses persisted at high levels up to the time of challenge at 18 weeks postimmunization, and responding cells maintained an effector memory phenotype. Despite the ability of the RRVenv recombinant to express high levels of Env in cultured cells, and despite the appearance of strong anti-RRV antibody responses in immunized monkeys, anti-Env antibody responses were below our ability to detect them. Immunized monkeys, together with three unimmunized controls, were challenged intravenously with 10 monkey infectious doses of SIVmac239. All five immunized monkeys and all three controls became infected with SIV, but peak viral loads were 1.2 to 3.0 log(10) units lower and chronic-phase viral loads were 1.0 to 3.0 log(10) units lower in immunized animals than the geometric mean of unimmunized controls. These differences were statistically significant. Anti-Env antibody responses following challenge indicated an anamnestic response in the vaccinated monkeys. These findings further demonstrate the potential of recombinant herpesviruses as preventive vaccines for AIDS. We hypothesize that this live, replication-competent, persistent herpesvirus vector could match, or come close to matching, live attenuated strains of SIV in the degree of protection if the difficulty with elicitation of anti-Env antibody responses can be overcome.

Antigen-specific T cell responses to BK polyomavirus antigens identify functional anti-viral immunity and may help to guide immunosuppression following renal transplantation

Infection with the polyoma virus BK (BKV) is a major cause of morbidity following renal transplantation. Limited understanding of the anti-viral immune response has prevented the design of a strategy that balances treatment with the preservation of graft function. The proven utility of interferon-gamma enzyme-linked immunospot (ELISPOT) assays to measure T cell responses in immunocompetent hosts was the basis for trying to develop a rational approach to the management of BKV following renal transplantation. In a sample of transplant recipients and healthy controls, comparisons were made between T cell responses to the complete panel of BKV antigens, the Epstein-Barr virus (EBV) antigens, BZLF1 and EBNA1, and the mitogen phytohaemagglutinin (PHA). Correlations between responses to individual antigens and immunosuppressive regimens were also analysed. Antigen-specific T cell responses were a specific indicator of recent or ongoing recovery from BKV infection (P < 0·05), with responses to different BKV antigens being highly heterogeneous. Significant BKV immunity was undetectable in transplant patients with persistent viral replication or no history of BKV reactivation. Responses to EBV antigens and mitogen were reduced in patients with BKV reactivation, but these differences were not statistically significant. The T cell response to BKV antigens is a useful and specific guide to recovery from BKV reactivation in renal transplant recipients, provided that the full range of antigenic responses is measured.

Evaluation of Oral Immunization with Recombinant Avian Influenza Virus HA1 Displayed on the Lactococcus lactis Surface and Combined with the Mucosal Adjuvant Cholera Toxin Subunit B

The development of safe and efficient avian influenza vaccines for human and animal uses is essential for preventing virulent outbreaks and pandemics worldwide. In this study, we constructed a recombinant (pgsA-HA1 gene fusion) Lactococcus lactis strain that expresses and displays the avian influenza virus HA1 antigens on its surface. The vectors were administered by oral delivery with or without the addition of cholera toxin subunit B (CTB). The resulting immune responses were analyzed, and the mice were eventually challenged with lethal doses of H5N1 viruses. Significant titers of hemagglutinin (HA)-specific serum IgG and fecal IgA were detected in the group that also received CTB. Cellular immunities were also shown in both cell proliferation and gamma interferon (IFN-γ) enzyme-linked immunospot (ELISpot) assays. Most importantly, the mice that received the L. lactis pgsA-HA1 strain combined with CTB were completely protected from lethal challenge of the H5N1 virus. These findings support the further development of L. lactis-based avian influenza virus vaccines for human and animal uses.

Safety and immunogenicity of heat-treated zoster vaccine (ZVHT) in adults with solid tumor malignancy (STM).

2551 Background: Herpes zoster incidence is higher in patients undergoing chemotherapy for solid tumor malignancy [STM] (12-15 cases/1000 person-years) than the general population (~3-5 cases/1000 person-years). A heat-treated zoster vaccine [ZVHT] was assessed in adults with STM. Methods: Randomized, double-blind, placebo-controlled, multicenter Phase I study of 4 doses of ZVHT or placebo [Pbo] (~30 days apart) in adults ≥18 years with STM. Of 79 subjects enrolled, 59 received ZVHT and 20 Pbo. All vaccinated patients evaluated for adverse events [AEs] through day 28 postdose 4. Blood collected at baseline & ~28 days after each dose to measure (1) VZV antibody concentrations by glycoprotein enzyme-linked immunosorbent assay [gpELISA], and (2) varicella zoster virus [VZV] T-cell responses by interferon-gamma enzyme-linked immunospot (IFN-γ ELISPOT) assay. Primary hypothesis: 4 doses of ZVHT would elicit a statistically significant VZV-specific immune response (lower bound 90% CI on geometric mean fold rise [GMFR] >1.0) in adults with STM. Results: Injection-site AEs occurred in 36.8% of ZVHT group and 5.3% of Pbo group. No reported vaccine-related serious AEs. One AE resulting in death was reported in the ZVHT group (acute respiratory failure, 31 days postdose 1); deemed not vaccine-related by investigator. One ZVHT recipient discontinued due to a vaccine-related AE (injection site erythema, 1 day postdose 3). No ZVHT recipient had a rash that was PCR positive for the vaccine strain of VZV. At baseline in the ZVHT group, VZV-specific gpELISA geometric mean titer (GMT) was 235 (90% CI: 163, 338) and the IFN-γ ELISPOT geometric mean count (GMC) was 24 (90% CI: 16, 37). Both measures increased Postdose 4: gpELISA GMT was 452 (90% CI: 342, 597); IFN-γ ELISPOT GMC was 89 (90% CI: 57, 138). The primary hypothesis was met, as Postdose 4 immune responses in the ZVHT group were significantly higher than baseline for both gpELISA (GMFR=2.4 [90% CI: 1.8, 3.0], p<0.0001) and IFN-γ ELISPOT (GMFR=3.0 [90% CI: 2.0, 4.6], p<0.0001). Conclusions: In the STM population, ZVHT had an acceptable safety profile and elicited statistically significant VZV-specific antibody and functional T-cell responses at 28 days Postdose 4.

Transcriptionally Abundant Major Histocompatibility Complex Class I Alleles Are Fundamental to Nonhuman Primate Simian Immunodeficiency Virus-Specific CD8+T Cell Responses

Simian immunodeficiency virus (SIV)-infected macaques are the preferred animal model for human immunodeficiency virus (HIV) vaccines that elicit CD8(+) T cell responses. Unlike humans, whose CD8(+) T cell responses are restricted by a maximum of six HLA class I alleles, macaques express up to 20 distinct major histocompatibility complex class I (MHC-I) sequences. Interestingly, only a subset of macaque MHC-I sequences are transcriptionally abundant in peripheral blood lymphocytes. We hypothesized that highly transcribed MHC-I sequences are principally responsible for restricting SIV-specific CD8(+) T cell responses. To examine this hypothesis, we measured SIV-specific CD8(+) T cell responses in MHC-I homozygous Mauritian cynomolgus macaques. Each of eight CD8(+) T cell responses defined by full-proteome gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay were restricted by four of the five transcripts that are transcriptionally abundant (>1% of total MHC-I transcripts in peripheral blood lymphocytes). The five transcriptionally rare transcripts shared by these animals did not restrict any detectable CD8(+) T cell responses. Further, seven CD8(+) T cell responses were defined by identifying peptide binding motifs of the three most frequent MHC-I transcripts on the M3 haplotype. Combined, these results suggest that transcriptionally abundant MHC-I transcripts are principally responsible for restricting SIV-specific CD8(+) T cell responses. Thus, only a subset of the thousands of known MHC-I alleles in macaques should be prioritized for CD8(+) T cell epitope characterization.

Safety and Immunogenicity of Heat-Treated Zoster Vaccine (ZVHT) In Adults with Hematologic Malignancy (HM).

AbstractAbstract 1394 BackgroundHerpes zoster incidence is higher in patients undergoing chemotherapy for hematologic malignancy [HM] (25-100 cases/1000 person-years) than in the general population (≂f3-5 cases/1000 person-years) and the general older adult population (7-12 cases/1000 person-years). The licensed live-attenuated zoster vaccine was heat-treated [ZVHT] and assessed in adults with HM. MethodsRandomized, double-blind, placebo-controlled, multicenter Phase I study of 4 ZVHT doses (≂f30 days apart) in adults ≥18 years with HM. Eighty subjects were enrolled, of whom 61 received ZVHT and 19 placebo (Pbo). Blood was collected at baseline and ≂f28 days after each dose to measure (1) VZV antibody concentrations by glycoprotein enzyme-linked immunosorbent assay (gpELISA), and (2) varicella zoster virus (VZV) T-cell responses by interferon-gamma enzyme-linked immunospot (IFN-γ ELISPOT) assay. All vaccinated patients were evaluated for adverse events (AEs) through day 28 postdose 4.The primary hypothesis was that 4 doses of ZVHT would elicit a statistically significant VZV-specific immune response (lower bound of 90% CI on geometric mean fold rise [GMFR] >1.0) in adults with HM. ResultsInjection-site adverse events (AEs) occurred in 31.1% of the ZVHT group and 10.5% of the Pbo group. Of overall vaccine-related systemic AEs (ZVHT: 18.0%, Pbo: 15.8%), only nausea was reported by >10% in either group (ZVHT: 0.0%, Pbo: 10.5%). There were 2 reported vaccine-related serious AEs in the ZVHT group (vomiting, 2 days postdose 1; peripheral neuropathy, 6 days postdose 2). Four AEs resulting in death were reported in the ZVHT group (coronary arteriosclerosis, 24 days postdose 1; malignant neoplasm, 18 days postdose 2; leukemia, 10 days postdose 2; neutropenic sepsis, 48 days postdose 2), all deemed not vaccine-related by the investigator. Two subjects discontinued due to vaccine-related AEs (peripheral neuropathy, ZVHT 6 days postdose 2; nausea and vomiting, Pbo 2 days postdose 1). No ZVHT recipient had a rash that was PCR positive for the vaccine strain of VZV.At baseline, the VZV-specific gpELISA GMT was 205 (90% CI: 158, 267) and the IFN-γ ELISPOT GMC was 11 (90% CI: 7, 18). Both measures increased Postdose 4: gpELISA GMT was 262 (90% CI: 204, 336); IFN-γ ELISPOT GMC was 24 (90% CI: 14, 41). The primary hypothesis was met, as Postdose 4 immune responses were significantly higher than baseline for both gpELISA (GMFR=1.3 [90% CI: 1.1, 1.5], p=0.003) and IFN-γ ELISPOT (GMFR= 2.2 [90% CI: 1.4, 3.5], p=0.004). ConclusionIn the HM population, ZVHT had an acceptable safety profile and elicited statistically significant VZV-specific antibody and functional T-cell responses at 28 days Postdose 4. Disclosures:Camacho:Merck: Research Funding. Off Label Use: This clinical research study examined the safety and immunogenicity of a heat-treated version of the licensed live-attenuated zoster vaccine (Zostavax) assessed in adults with hematologic malignancy. Betts:Merck: Research Funding. Wertheim:Merck: Research Funding. Zhao:Merck: Employment. Fernsler:Merck: Employment. Manoff:Merck: Employment. Annunziato:Merck: Employment.

Failure to Detect Xenotropic Murine Leukemia Virus–Related Virus in Blood of Individuals at High Risk of Blood‐Borne Viral Infections

A xenotropic murine leukemia virus-related virus (XMRV) has recently been reported in association with prostate cancer and chronic fatigue syndrome, with a prevalence of up to 3.7% in the healthy population. We looked for XMRV in 230 patients with human immunodeficiency virus type 1 or hepatitis C infection. XMRV was undetectable in plasma or peripheral blood mononuclear cells by polymerase chain reaction targeting XMRV gag or env. T cell responses to XMRV Gag were undetectable in peripheral blood mononuclear cells by ex vivo gamma interferon enzyme-linked immunospot assay. In our cohorts, XMRV was not enriched in patients with blood-borne or sexually transmitted infections from the United Kingdom and Western Europe.

Chronic Graft Versus Host Disease Is Associated With an Immune Response to Autologous Human Leukocyte Antigen–Derived Peptides

Chronic graft-versus-host disease (cGVHD) is a major complication of allogeneic bone marrow transplantation (BMT) the immunopathogenesis of which is not well understood. Humoral and cellular immunity are both implicated, patients express a range of autoantibodies, but the targets of cellular immunity are not well defined. Autologous human leukocyte antigen (HLA)-derived peptides constitute a significant proportion of the repertoire. We have investigated the response to HLA-derived peptides after allogeneic BMT using gamma-interferon enzyme-linked immunospot assay (ELISPOT). We also studied the release of this gamma-interferon by flow cytometry in a subgroup of responsive patients. The peripheral blood mononuclear cell response was assessed by gamma-interferon ELISPOT in 42 BMT recipients (21 with cGVHD) and 30 healthy donors. Thirteen of 21 patients diagnosed with cGVHD responded to at least one HLA-derived peptide compared with 1 of 21 patients without cGVHD (62% vs. 5%, P<10) and 1 of 30 healthy donors. In all but one patient these peptides correspond with the sequences of autologous HLA. The median single peptide-specific response in ELISPOT was 43/10 peripheral blood mononuclear cells. In a subgroup studied by flow cytometry, gamma-interferon production to individual peptides occurred in 0.04% to 0.18% of CD4 T lymphocytes. These observations identify HLA-derived peptides as targets of a cellular immune response in cGVHD.

Fluidity of HIV-1-Specific T-Cell Responses during Acute and Early Subtype C HIV-1 Infection and Associations with Early Disease Progression

Deciphering immune events during early stages of human immunodeficiency virus type 1 (HIV-1) infection is critical for understanding the course of disease. We characterized the hierarchy of HIV-1-specific T-cell gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay responses during acute subtype C infection in 53 individuals and associated temporal patterns of responses with disease progression in the first 12 months. There was a diverse pattern of T-cell recognition across the proteome, with the recognition of Nef being immunodominant as early as 3 weeks postinfection. Over the first 6 months, we found that there was a 23% chance of an increased response to Nef for every week postinfection (P = 0.0024), followed by a nonsignificant increase to Pol (4.6%) and Gag (3.2%). Responses to Env and regulatory proteins appeared to remain stable. Three temporal patterns of HIV-specific T-cell responses could be distinguished: persistent, lost, or new. The proportion of persistent T-cell responses was significantly lower (P = 0.0037) in individuals defined as rapid progressors than in those progressing slowly and who controlled viremia. Almost 90% of lost T-cell responses were coincidental with autologous viral epitope escape. Regression analysis between the time to fixed viral escape and lost T-cell responses (r = 0.61; P = 0.019) showed a mean delay of 14 weeks after viral escape. Collectively, T-cell epitope recognition is not a static event, and temporal patterns of IFN-γ-based responses exist. This is due partly to viral sequence variation but also to the recognition of invariant viral epitopes that leads to waves of persistent T-cell immunity, which appears to associate with slower disease progression in the first year of infection.

Evaluation of Cytomegalovirus (CMV)–Specific T Cell Immune Reconstitution Revealed That Baseline Antiviral Immunity, Prophylaxis, or Preemptive Therapy but not Antithymocyte Globulin Treatment Contribute to CMV‐Specific T Cell Reconstitution in Kidney Transplant Recipients

The ultimate goal of organ transplantation is the reestablishment of organ function and the restoration of a solid immunity to prevent the assault of potentially deadly pathogens. T cell immunity is crucial in controlling cytomegalovirus (CMV) infection. It is still unknown how preexisting antiviral T cell levels, prophylaxis, or preemptive antiviral strategies and pharmacological conditioning affect immune reconstitution. Seventy preemptively treated CMV-seropositive recipients, 13 prophylaxis-treated CMV-seronegative recipients of seropositive donor transplants, 2 seropositive recipients of seronegative donor kidneys, and 27 pretransplant subjects were enrolled in a cross-sectional study and analyzed for CMV viremia (DNAemia) and CMV-specific T cell response (interferon-gamma enzyme-linked immunospot assay) before transplantation and at 30, 60, 90, 180, and 360 days after transplantation. CMV-seropositive transplant recipients displayed a progressive but heterogeneous pattern of immune reconstitution starting from day 60 after transplantation. CMV-seronegative recipients did not mount a detectable T cell response throughout the prophylaxis regimen. A single episode of CMV viremia (CMV copy number, 7000-170,000 copies/mL) was sufficient to prime a protective T cell immune response in CMV-seronegative recipients. Antithymocyte globulin treatment did not significantly affect CMV-specific T cell response. Baseline immunity, antiviral therapy but not antithymocyte globulin treatments profoundly influence T cell reconstitution in kidney transplant recipients.

T-Cell Response to Human Papillomavirus Type 58 L1, E6, and E7 Peptides in Women with Cleared Infection, Cervical Intraepithelial Neoplasia, or Invasive Cancer

Human papillomavirus type 58 (HPV-58) exists in a relatively high prevalence in certain parts of the world, including East Asia. This study examined the T-cell response to HPV-58 L1, E6, and E7 peptides among women with cleared infection, cervical intraepithelial neoplasia grade 2 (CIN2) or CIN3, or invasive cervical cancer (ICC). Peptides found to be reactive in the in vitro peptide binding assay or mouse-stimulating study were tested with a gamma interferon (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay to detect peptide-specific responses from the peripheral blood mononuclear cells (PBMC) collected from 91 HPV-58-infected women (32 with cleared infection, 16 CIN2, 15 CIN3, and 28 ICC). Four HLA-A11-restricted HPV-58 L1 peptides, located at amino acid positions 296 to 304, 327 to 335, 101 to 109, and 469 to 477, showed positive IFN-gamma ELISPOT results and were mainly from women with cleared infection. Two HLA-A11-restricted E6 peptides (amino acid positions 64 to 72 and 94 to 102) and three HLA-A11-restricted E7 peptides (amino acid positions 78 to 86, 74 to 82, and 88 to 96) showed a positive response. A response to E6 and E7 peptides was mainly observed from subjects with CIN2 or above. One HLA-A2-restricted E6 peptide, located at amino acid position 99 to 107, elicited a positive response in two CIN2 subjects. One HLA-A24-restricted L1 peptide, located at amino acid position 468 to 476, also elicited a positive response in two CIN2 subjects. In summary, this study has identified a few immunogenic epitopes for HPV-58 E6 and E7 proteins. It is worthwhile to further investigate whether responses to these epitopes have a role in clearing an established cervical lesion.

Diagnostic usefulness of enzyme-linked immunospot assay for interferon-gamma for tuberculosis in cancer patients

The interferon-γ enzyme-linked immunospot assay (ELISPOT) has been demonstrated to be useful in the diagnosis of active tuberculosis (TB). In this study we aimed to evaluate the diagnostic performance of the ELISPOT assay in cancer patients with suspected pulmonary TB. Eighty-one cancer patients with suspected pulmonary TB were prospectively enrolled from April 2007 to December 2008, to investigate the diagnostic sensitivity and specificity of the ELISPOT assay. Of the 38 patients with TB, 33 (86.8%) had positive ELISPOT results. Of the 43 patients without TB, the results of the ELISPOT assay were negative in 35 (81.3%) patients. The overall sensitivity was 86.8%, specificity 81.3%, positive predictive value 80.5% and negative predictive value 87.5%. No significant difference was noted for the diagnostic performance of the ELISPOT assay for diagnosing TB between solid cancer and haematological cancer patients. In addition, a quantitative study did not show that TB patients with solid cancers have a better response than haematological cancer patients as measured by spot-forming cells per 10(6) peripheral blood mononuclear cells after exposure to early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10). In conclusion, the ELISPOT assay could be a useful supplementary tool for the diagnosis of pulmonary TB among cancer patients, irrespective of cancer type.

Enzyme-linked immunospot assay for interferon-gamma to support the diagnosis of tuberculosis in diabetic patients

The aim of this study was to evaluate the support to tuberculosis diagnosis of a new immunological tool, an enzyme-linked immunospot (ELISPOT) assay for interferon-γ, in diabetic patients. From March 2007 to January 2008, diabetic patients with suspected pulmonary tuberculosis were enrolled in a tertiary care hospital. Data on clinical characteristics of the patients and conventional laboratory results were collected and blood samples were obtained for ELISPOT assay (T SPOT-TB; Oxford Immunotec Ltd, Oxford, UK). A total of 84 patients with suspected pulmonary tuberculosis were recruited. Fifty-one (60.7%) of these patients were considered to have pulmonary tuberculosis, including 42 (50%) with confirmed tuberculosis and 9 (10.7%) with probable tuberculosis. The overall (confirmed and probable tuberculosis) sensitivity, specificity, positive predictive value and negative predictive value of the ELISPOT assay were 84.3%, 66.7%, 79.6%, and 73.3%, respectively. The negative predictive value of the ELISPOT assay was significantly higher in patients with adequate glycaemic control (90% vs 56.3%). In conclusion, the ELISPOT assay can provide useful support in diagnosing pulmonary tuberculosis in diabetic patients, especially those with adequate glycaemic control.

Gentamicin‐induced readthrough of stop codons in duchenne muscular dystrophy

The objective of this study was to establish the feasibility of long-term gentamicin dosing to achieve stop codon readthrough and produce full-length dystrophin. Mutation suppression of stop codons, successfully achieved in the mdx mouse using gentamicin, represents an important evolving treatment strategy in Duchenne muscular dystrophy (DMD). Two DMD cohorts received 14-day gentamicin (7.5mg/kg/day): Cohort 1 (n = 10) stop codon patients and Cohort 2 (n = 8) frameshift controls. Two additional stop codon DMD cohorts were gentamicin treated (7.5mg/kg) for 6 months: Cohort 3 (n = 12) dosed weekly and Cohort 4 (n = 4) dosed twice weekly. Pre- and post-treatment biopsies were assessed for dystrophin levels, as were clinical outcomes. In the 14-day study, serum creatine kinase (CK) dropped by 50%, which was not seen in frameshift DMD controls. After 6 months of gentamicin, dystrophin levels significantly increased (p = 0.027); the highest levels reached 13 to 15% of normal (1 in Cohort 3, and 2 in Cohort 4), accompanied by reduced serum CK favoring drug-induced readthrough of stop codons. This was supported by stabilization of strength and a slight increase in forced vital capacity. Pretreatment stable transcripts predicted an increase of dystrophin after gentamicin. Readthrough efficiency was not affected by the stop codon or its surrounding fourth nucleotide. In 1 subject, antigen-specific interferon-gamma enzyme-linked immunospot assay detected an immunogenic dystrophin epitope. The results support efforts to achieve drug-induced mutation suppression of stop codons. The immunogenic epitope resulting from readthrough emphasizes the importance of monitoring T-cell immunity during clinical studies that suppress stop codons. Similar principles apply to other molecular strategies, including exon skipping and gene therapy.

Safety and Immunogenicity of Modified Vaccinia Ankara (ACAM3000): Effect of Dose and Route of Administration

We conducted a clinical trial of the safety and immunogenicity of modified vaccinia Ankara (MVA) to examine the effects of dose and route of administration. Seventy-two healthy, vaccinia virus-naive subjects received 1 of 6 regimens of MVA (ACAM3000) or placebo consisting of 2 administrations given 1 month apart. MVA was generally well tolerated at all dose levels and by all routes. More pronounced local reactogenicity was seen with the intradermal and subcutaneous routes than with intramuscular administration. Binding antibodies to whole virus and neutralizing antibodies to the intracellular mature virion and extracellular enveloped virion forms of vaccinia virus were elicited by all routes of MVA administration and were greater for the higher dose by each route. Similar levels of neutralizing antibodies were seen at a 10-fold-lower dose given intradermally (1 x 10(7) median tissue culture infective doses [TCID(50)]), compared with responses after 1 x 10(8) TCID(50) given intramuscularly or subcutaneously. T cell immune responses to vaccinia virus were detected by an interferon gamma enzyme-linked immunospot assay but had no clear relationship to dose or route. These data suggest that intradermal immunization with MVA provides a dose-sparing effect by eliciting antibody responses similar in magnitude and kinetics to those elicited by the intramuscular or subcutaneous routes but at a 10-fold-lower dose.

An Epitope of Bacillus anthracis Protective Antigen That Is Cryptic in Rabbits May Be Immunodominant in Humans

In a recent article, Oscherwitz et al. endeavor to enhance the immunogenicity of a multiple antigenic peptide (MAP) vaccine that targets a loop-neutralizing determinant (LND) of Bacillus anthracis protective antigen (PA) (1). They showed in a previous study (2) that this MAP, consisting of four copies of amino acids 305 to 319 of PA (PA 305-319) extending from a lysine core, can elicit humoral immunity in rabbits that is specific and strongly neutralizing for the 2β2-2β3 loop in domain 2 of PA. However, LND-specific antibodies were not detected in rabbits immunized with whole PA, and it was concluded that PA 305-319 may be a cryptic epitope. T cell assays in PA 305-319-immunized mice showed that this peptide appeared to lack activity as a T helper cell epitope. We have good evidence that humans exposed to PA, either following cutaneous anthrax infection or through vaccination, generate long-term, T cell memory to both whole PA and PA 305-319. In a study including samples from naturally exposed patients recovered from cutaneous anthrax, from vaccine-hyperimmunized AVP (anthrax vaccine precipitated) donors, and from recombinant PA-vaccinated donors, positive T cell gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay responses were detected across all groups on restimulation of peripheral blood mononuclear cells (PBMC) with both whole PA and PA 305-319 (Table 1; unpublished data). This shows that humans exposed to B. anthracis and whole PA recognize and generate memory to PA 305-319, suggesting that it is immunodominant rather than cryptic. Donors expressed a range of HLA haplotypes, with a majority being HLA-DR4 positive. While many important discoveries on the immunology of infection are made in experimental models, the human T cell response to PA in this respect differs from that of both rabbits and mice. In this light, the epitope used by Oscherwitz and colleagues may indeed be more relevant to eliciting appropriate human immunity than previously envisaged.

Enhanced Anti-HIV Functional Activity Associated with Gag-Specific CD8 T-Cell Responses

Effective HIV-specific T-cell immunity requires the ability to inhibit virus replication in the infected host, but the functional characteristics of cells able to mediate this effect are not well defined. Since Gag-specific CD8 T cells have repeatedly been associated with lower viremia, we examined the influence of Gag specificity on the ability of unstimulated CD8 T cells from chronically infected persons to inhibit virus replication in autologous CD4 T cells. Persons with broad (>or=6; n = 13) or narrow (<or=1; n = 13) Gag-specific responses, as assessed by gamma interferon enzyme-linked immunospot assay, were selected from 288 highly active antiretroviral therapy (HAART)-naive HIV-1 clade C-infected South Africans, matching groups for total magnitude of HIV-specific CD8 T-cell responses and CD4 T-cell counts. CD8 T cells from high Gag responders suppressed in vitro replication of a heterologous HIV strain in autologous CD4 cells more potently than did those from low Gag responders (P < 0.003) and were associated with lower viral loads in vivo (P < 0.002). As previously shown in subjects with low viremia, CD8 T cells from high Gag responders exhibited a more polyfunctional cytokine profile and a stronger ability to proliferate in response to HIV stimulation than did low Gag responders, which mainly exhibited monofunctional CD8 T-cell responses. Furthermore, increased polyfunctionality was significantly correlated with greater inhibition of viral replication in vitro. These data indicate that enhanced suppression of HIV replication is associated with broader targeting of Gag. We conclude that it is not the overall magnitude but rather the breadth, magnitude, and functional capacity of CD8 T-cell responses to certain conserved proteins, like Gag, which predict effective antiviral HIV-specific CD8 T-cell function.

Extralymphoid CD8+ T cells resident in tissue from simian immunodeficiency virus SIVmac239{Delta}nef-vaccinated macaques suppress SIVmac239 replication ex vivo.

Live-attenuated vaccination with simian immunodeficiency virus (SIV) SIVmac239Deltanef is the most successful vaccine product tested to date in macaques. However, the mechanisms that explain the efficacy of this vaccine remain largely unknown. We utilized an ex vivo viral suppression assay to assess the quality of the immune response in SIVmac239Deltanef-immunized animals. Using major histocompatibility complex-matched Mauritian cynomolgus macaques, we did not detect SIV-specific functional immune responses in the blood by gamma interferon (IFN-gamma) enzyme-linked immunospot assay at select time points; however, we found that lung CD8(+) T cells, unlike blood CD8(+) T cells, effectively suppress virus replication by up to 80%. These results suggest that SIVmac239Deltanef may be an effective vaccine because it elicits functional immunity at mucosal sites. Moreover, these results underscore the limitations of relying on immunological measurements from peripheral blood lymphocytes in studies of protective immunity to HIV/SIV.

Relationship of Reconstituted Adaptive and Innate Cytomegalovirus (CMV)–Specific Immune Responses with CMV Viremia in Hematopoietic Stem Cell Transplant Recipients

Cytomegalovirus (CMV) is a major cause of morbidity in transplant recipients. An immunologic predictor of protection against CMV reactivation or disease is highly desirable. Thirty-eight allogeneic hematopoietic stem cell transplant recipients at risk of CMV disease were prospectively monitored using whole blood CMV-DNA polymerase chain reaction assay, lymphocyte proliferation assay (LPA), interferon gamma enzyme-linked immunospot assay (ELISPOT), and flow cytometric enumeration of CMV-specific CD69+interferon (IFN)gamma+CD4, CD8, natural killer cells, and gammadelta T cells. Twenty-one subjects developed > or =1 episode of CMV viremia and 4 developed disease during 360 days of follow-up. Among CMV-seropositive recipients, positive CMV-LPA before transplantation correlated with higher risk of developing viremia after transplantation (P = .02). In contrast, after transplantation, reconstitution of CMV-LPA was significantly associated with absence of CMV viremia over 360 days of follow-up (P = .04) and with faster clearance of viremia during individual episodes of CMV reactivation (P = .03). Reconstitution of CMV-specific natural killer cells was also associated with absence of CMV viremia over 360 days of study (P = .04) but not with faster clearance of viremia. CMV-specific CD4, CD8, gammadelta T cells, and ELISPOT values were not significantly different in viremic subjects, compared with the corresponding values in nonviremic subjects, at any time point. To our knowledge, this is the first study to prospectively compare multiple measures of innate and adaptive immune responses in hematopoietic stem cell transplant recipients with CMV viremia. The strongest immune correlates with protection against CMV viremia in hematopoietic stem cell transplant recipients are reconstitution of CMV-specific T cell memory responses (LPA) and recovery of natural killer cell function. In contrast, positive CMV-LPA before transplantation may be a marker of high risk of CMV reactivation after transplantation.

Strong CD8+ T-cell responses against tumor-associated antigens prolong the recurrence-free interval after tumor treatment in patients with hepatocellular carcinoma

We investigated whether tumor-specific CD8(+) T-cell responses affect tumor-free survival as well as the relationship between CD8(+) T-cell responses against tumor-associated antigens (TAAs) and the clinical course after tumor treatment in patients with hepatocellular carcinoma (HCC). Twenty patients with HCC that were treated by radiofrequency ablation or trans-catheter chemo-embolization (TACE) and in whom HCC was undetectable by ultrasonography, CT, and/or MRI 1 month after treatment were enrolled in the study. Before and after treatment for HCC, analyses of TAA (glypican-3, NY-ESO-1, and MAGE-1)-specific CD8(+) T-cell responses were evaluated with an interferon-gamma enzyme-linked immunospot (ELISpot) assay using peripheral CD8(+) T-cells, monocytes, and 104 types of 20-mer synthetic peptide overlapping by 10 residues and spanning the entirety of the 3 TAAs. Sixteen out of 20 patients (80%) showed a positive response (> or = 10 TAA-specific cells/10(5) CD8(+) T-cells) before or after treatment. When we performed univariate analysis of prognostic factors for the tumor-free period in the 20 patients, platelet count, prothrombin time, and the number of TAA-specific CD8(+) T-cells after treatment were significant factors (P = 0.027, 0.030, and 0.004, respectively). In multivariate analysis, the magnitude of the TAA-specific CD8(+) T-cell response (> or = 40 TAA-specific cells/10(5) CD8(+) T-cells) was the only significant prognostic factor for a prolonged tumor-free interval (hazard ratio 0.342, P = 0.022). Our results suggest that strong TAA-specific CD8(+) T-cell responses suppress the recurrence of HCC. Immunotherapy to induce TAA-specific cytotoxic T lymphocytes by means such as the use of peptide vaccines should be considered for clinical application in patients with HCC after local therapy.