Biomedical subjects

Helen Horton

Publications and source records attributed to Helen Horton.

17 recordsLinked to original sources

PubMed2007

Phase 2 study of an HIV-1 canarypox vaccine (vCP1452) alone and in combination with rgp120: negative results fail to trigger a phase 3 correlates trial.

BACKGROUND: A goal of T-cell HIV vaccines is to define the correlation between a vaccine-induced immune response and protection from HIV infection. We conducted a phase 2 trial to determine if a canarypox vaccine candidate (vCP1452) administered with rgp120 subunit protein would "qualify" for a trial to define a correlate of efficacy. METHODS: A total of 330 healthy volunteers were enrolled into 4 groups: 120 received vCP1452 alone (0, 1, 3, and 6 months), 120 received vCP1452 with 2 different regimens of rgp120 coadministration, and 90 received placebo. HIV-specific antibody responses were measured by enzyme-linked immunoassay (ELISA) and neutralizing activity. T-cell responses were measured by chromium release and interferon-gamma (IFNgamma) enzyme-linked immunospot (ELISpot) assay. RESULTS: Significant neutralizing antibody responses to the HIV MN strain were detected in all vaccine groups, with net responses ranging from 57% (95% confidence interval [CI]: 40% to 71%) to 94% (95% CI: 85% to 99%). Net cumulative HIV-specific CD8 IFNgamma ELISpot assay responses were 13% (95% CI: -1% to 26%) for recipients of vCP1452 alone and 16% (95% CI: 2% to 29%) for recipients of vCP1452 plus rgp120. CONCLUSIONS: Overall, the HIV-specific CD8 cytotoxic T lymphocyte (CTL) response was not sufficient to qualify the regimen for a subsequent trial designed to detect an immune correlate of protection requiring a minimum CD8 CTL frequency of 30%.

AIDS Vaccines↗

PubMed2006

Does preserving memory correlate with surviving HIV?

The holy grail for HIV-1 vaccine researchers is to develop an efficacious vaccine, a goal that would be aided by defining a correlate of protection from infection. Recently, the authors of two papers have come one step closer to this goal with the definition of a correlate of survival following simian immunodeficiency virus (SIV) infection: the preservation of memory CD4(+) T cells during acute infection. Both of these research groups show that vaccination can prevent the initial immune devastation caused by SIV infection and that this correlates with survival following SIV challenge. Here, we highlight the significance of these two recent papers in light of the urgent need to produce an efficacious HIV vaccine and discuss several crucial issues that must be addressed before the correlate can be used in human clinical trials.

Animals↗

PubMed2006

Preservation of T cell proliferation restricted by protective HLA alleles is critical for immune control of HIV-1 infection.

HIV-1-infected persons with HLA-B27 and -B57 alleles commonly remain healthy for decades without antiretroviral therapy. Properties of CD8+ T cells restricted by these alleles considered to confer disease protection in these individuals are elusive but important to understand and potentially elicit by vaccination. To address this, we compared CD8+ T cell function induced by HIV-1 immunogens and natural infection using polychromatic flow cytometry. HIV-1-specific CD8+ T cells from all four uninfected immunized and 21 infected subjects secreted IFN-gamma and TNF-alpha. However, CD8+ T cells induced by vaccination and primary infection, but not chronic infection, proliferated to their cognate epitopes. Notably, B27- and B57-restricted CD8+ T cells from nonprogressors exhibited greater expansion than those restricted by other alleles. Hence, CD8+ T cells restricted by certain protective alleles can resist replicative defects, which permits expansion and antiviral effector activities. Our findings suggest that the capacity to maintain CD8+ T cell proliferation, regardless of MHC-restriction, may serve as an important correlate of disease protection in the event of infection following vaccination.

AIDS Vaccines↗

PubMed2006

Durable HIV-1 antibody and T-cell responses elicited by an adjuvanted multi-protein recombinant vaccine in uninfected human volunteers.

BACKGROUND: Use of the recombinant proteins NefTat and gp120(W61D) formulated with the AS02A adjuvant system was previously shown to protect against AIDS in a rhesus macaque SHIV animal model system. METHODS: Eighty-four HIV uninfected human participants were vaccinated intramuscularly at 0, 1, and 3 months and evaluated for safety. Immune responses were analyzed for the presence of vaccine-induced antibody and T lymphocyte responses. RESULTS: The vaccines were safe and well tolerated at all doses. Nef-, Tat-, and gp120-specific binding antibodies were induced in all individuals that received the respective antigen, lasting up to 9 months after the final immunization. Antibodies able to neutralize the T-cell laboratory-adapted strain of HIV-1(W61D) were detected in the majority of vacinees, but did not neutralize primary isolates. Envelope-specific antibody-dependent cell cytoxicity was detected in most of the individuals receiving gp120. Robust and persistent HIV-specific lymphoproliferative responses were detected against all subunit proteins in the majority of immunized participants. As expected, HIV-specific CD8 T-cell responses were not detected. CONCLUSIONS: Despite the lack of primary isolate neutralizing antibody induction, the observed high frequency and magnitude of other immune responses warrant further work with this vaccine or vaccine components.

AIDS Vaccines↗

PubMed2006

Interleukin-2 reconstitutes defective human immunodeficiency virus (HIV), and cytomegalovirus (CMV) specific CD8+ T cell proliferation in HIV infection.

Recent studies indicate that a defective proliferative response of HIV-specific CD8+ T cells is associated with the lack of virologic control in chronic HIV infection in humans. The possible mechanisms that might be responsible for the reduced proliferative potential of HIV-specific CD8+ T cells and conditions conducive to the proliferation of CD8+ T cells were examined in 14 HIV-infected individuals and 7 HIV-uninfected controls using CFSE labeling and flow cytometry techniques, and analyzed data using 2 quantitative measurements: the percentages of proliferating CD8+ T cells (Tp), and the maximum number of cell divisions (Dm) after stimulation. It was found that CD8+ T cells from HIV-infected and -uninfected subjects proliferated equally well after polyclonal stimulation by phylohemagglutinin A (PHA); both groups reached a Tp of 92%-96% and a Dm of 5-8. However, in HIV-infected subjects, proliferation of HIV- and CMV-specific CD8+ T cells was significantly reduced compared to proliferation of CMV- specific CD8+ T cells from HIV-uninfected subjects. These defective proliferative responses of HIV- and CMV-specific CD8+ T cells were restored by the addition of IL-2 at the time of stimulation. These results may have implications for the design of immune modulation strategies in vivo.

Amino Acid Sequence↗

PubMed2006

Induction of human immunodeficiency virus type 1 (HIV-1)-specific T-cell responses in HIV vaccine trial participants who subsequently acquire HIV-1 infection.

Candidate human immunodeficiency virus type 1 (HIV-1) vaccines designed to elicit T-cell immunity in HIV-1-uninfected persons are under investigation in phase I to III clinical trials. Little is known about how these vaccines impact the immunologic response postinfection in persons who break through despite vaccination. Here, we describe the first comprehensive characterization of HIV-specific T-cell immunity in vaccine study participants following breakthrough HIV-1 infection in comparison to 16 nonvaccinated subjects with primary HIV-1 infection. Whereas none of the 16 breakthrough infections possessed vaccine-induced HIV-1-specific T-cell responses preinfection, 85% of vaccinees and 86% of nonvaccinees with primary HIV-1 infection developed HIV-specific T-cell responses postinfection. Breakthrough subjects' T cells recognized 43 unique HIV-1 T-cell epitopes, of which 8 are newly described, and 25% were present in the vaccine. The frequencies of gamma interferon (IFN-gamma)-secreting cells recognizing epitopes within gene products that were and were not encoded by the vaccine were not different (P = 0.64), which suggests that responses were not anamnestic. Epitopes within Nef and Gag proteins were the most commonly recognized in both vaccinated and nonvaccinated infected subjects. One individual controlled viral replication without antiretroviral therapy and, notably, mounted a novel HIV-specific HLA-C14-restricted Gag LYNTVATL-specific T-cell response. Longitudinally, HIV-specific T cells in this individual were able to secrete IFN-gamma and tumor necrosis factor alpha, as well as proliferate and degranulate in response to their cognate antigenic peptides up to 5 years postinfection. In conclusion, a vaccinee's ability to mount an HIV-specific T-cell response postinfection is not compromised by previous immunization, since the CD8+ T-cell responses postinfection are similar to those seen in vaccine-naïve individuals. Finding an individual who is controlling infection highlights the importance of comprehensive studies of breakthrough infections in vaccine trials to determine whether host genetics/immune responses and/or viral characteristics are responsible for controlling viral replication.

AIDS Vaccines↗

PubMed2005

High-dose recombinant Canarypox vaccine expressing HIV-1 protein, in seronegative human subjects.

BACKGROUND: In clinical trials, canarypox ALVAC-human immunodeficiency virus (HIV) vaccines have been shown to elicit human HIV-specific cytotoxic T lymphocyte (CTL) responses in some but not all healthy uninfected adults.Methods. A clinical trial was conducted to examine whether the vaccine vCP1452 would elicit a greater HIV-specific CTL response when given at a dose of 10(8.0) TCID50 (60 participants) than when given at the regular dose, 10(7.26) TCID50 (40 participants); as a control, a placebo vaccine preparation also was administered (10 participants). RESULTS: Two weeks after the last vaccination in a series, HIV-specific CTL responses were not significantly different when measured by either chromium-release assay (8% and 16% in the high- and regular-dose recipients, respectively) or interferon- gamma ELISpot assay (8% and 15% in the high- and regular-dose recipients, respectively); moreover, recipients of the higher dose had greater local and systemic reactions (P<.001). CONCLUSIONS: High reactogenicity associated with an increased dose of vCP1452 negates the need for further evaluation of this strategy to boost the frequency of HIV-specific CTL response in seronegative human subjects. Development of highly immunogenic canarypox vectors requires further work to optimize vector and insert design, as well as novel ways to increase dosage and to reduce reactogenicity.

AIDS Vaccines↗

PubMed2005

Standardization of cytokine flow cytometry assays.

BACKGROUND: Cytokine flow cytometry (CFC) or intracellular cytokine staining (ICS) can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common platform by which to compare the immunogenicity of different vaccine candidates across multiple international organizations conducting clinical trials. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online). RESULTS: Three sample types (activated, fixed, and frozen whole blood; fresh whole blood; and cryopreserved PBMC) were shipped to various sites, where ICS assays using cytomegalovirus (CMV) pp65 peptide mix or control antigens were performed in parallel in 96-well plates. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Results ((CD4+)cytokine+ cells and (CD8+)cytokine+ cells) were determined by each site. Raw data were also sent to a central site for batch analysis with a dynamic gating template. Mean inter-laboratory coefficient of variation (C.V.) ranged from 17-44% depending upon the sample type and analysis method. Cryopreserved peripheral blood mononuclear cells (PBMC) yielded lower inter-lab C.V.'s than whole blood. Centralized analysis (using a dynamic gating template) reduced the inter-lab C.V. by 5-20%, depending upon the experiment. The inter-lab C.V. was lowest (18-24%) for samples with a mean of > 0.5% IFNgamma + T cells, and highest (57-82%) for samples with a mean of < 0.1% IFNgamma + cells. CONCLUSION: ICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision, which improves as the frequency of responding cells increases. Cryopreserved PBMC may yield slightly more consistent results than shipped whole blood. Analysis, particularly gating, is a significant source of variability, and can be reduced by centralized analysis and/or use of a standardized dynamic gating template. Use of pre-aliquoted lyophilized reagents for stimulation and staining can provide further standardization to these assays.

Blood Preservation↗

PubMed2004

Correlation between interferon- gamma secretion and cytotoxicity, in virus-specific memory T cells.

ELISpot and intracellular cytokine staining are replacing the traditional cytolytic ((51)Cr release) assay method in vaccine trials using human immunodeficiency virus (HIV)-1, and it is widely assumed that the number of interferon (IFN)- gamma -secreting T cells is a surrogate for the level of cytolytic activity. Thus, we sought to determine whether the detection of IFN- gamma in CD8(+) T cells correlates with cytolytic ability in vitro. In 29 (69.0%) of 42 HIV-1-seronegative immunocompetent individuals (22 unvaccinated and 20 vaccinated), virus-specific T cell responses recognizing cytomegalovirus, Epstein-Barr virus, and influenza and HIV-1 Gag epitopes were detected by at least 1 assay method (ELISpot, intracellular cytokine staining, and/or (51)Cr release), and 18 (62.1%) of these 29 demonstrated both IFN- gamma secretion and cytolysis. There was strong correlation between the results of IFN- gamma ELISpot and those of IFN- gamma intracellular cytokine staining ( rho =0.88) and between the results of (51)Cr release and those of intracellular cytokine staining ( rho =0.81); although the correlation is not absolute, intracellular cytokine staining can be used--and is superior to ELISpot--as a surrogate for cytolytic assays.

Antigens, Viral↗

PubMed2004

Statistical considerations for the design and analysis of the ELISpot assay in HIV-1 vaccine trials.

Effector T lymphocyte responses are considered critical for controlling human immunodeficiency virus type-1 (HIV-1) infection. The enzyme-linked immunospot (ELISpot) assay has emerged as a primary means of assessing HIV-specific T cell responses, and the development of objective methods that distinguish positive and negative ELISpot responses while properly controlling the rate of false positives is critical. In this paper, we consider several statistical methods that are helpful in defining such a positive criterion. Simulation results under a variety of scenarios suggest that a permutation-based criterion using a resampling adjustment for multiple comparisons yields the desired false positive rate while remaining competitive with other potential criteria in terms of sensitivity. These results also provide guidance on the effect of the number of experimental and negative control replicate wells on assay sensitivity. Application of different potential positive criteria using ELISpot assay results from IFN-gamma-secreting T cells of HIV-1 seropositive and seronegative donors confirmed several of the results obtained under simulation. Our findings support the application of statistically-based positive criteria such as the permutation-based resampling approach in assessing HIV vaccine-induced T cell responses. Moreover, the proposed methods have potential utility in related HIV immunopathogenesis studies and in non-HIV clinical vaccine trials.

AIDS Serodiagnosis↗

PubMed2002

Differences between T cell epitopes recognized after immunization and after infection.

Evidence suggests that cellular immune responses play a crucial role in the control of HIV and SIV replication in infected individuals. Several vaccine strategies have therefore targeted these CD8(+) and CD4(+) responses. Whether vaccination induces the same repertoire of responses seen after infection is, however, a key unanswered question in HIV vaccine development. We therefore compared the epitope specificity induced by vaccination to that present postchallenge in the peripheral blood. Intracellular cytokine staining of PBMC stimulated with overlapping 15/20-mer peptides spanning the proteins of SIV were measured after DNA/modified vaccinia Ankara vaccination of eight rhesus macaques. Lymphocytes from 8 animals recognized a total of 39 CD8 epitopes and 41 CD4 epitopes encoded by the vaccine. T cell responses were again monitored after challenge with SIVmac239 to investigate the evolution of these responses. Only 57% of all CD8(+) T cell responses and 19% of all CD4(+) T cell responses present after vaccination were recalled after infection as measured in the peripheral blood. Interestingly, 29 new CD8 epitopes and 5 new CD4 epitopes were recognized by PBMC in the acute phase. These new epitopes were not detected after vaccination, and only some of them were maintained in the chronic phase (33% of CD8 and no CD4 responses). Additionally, 24 new CD8 epitopes and 7 new CD4 epitopes were recognized by PBMC in the chronic phase of infection. The repertoire of the immune response detected in the peripheral blood after immunization substantially differed from the immune response detected in the peripheral blood after infection.

Administration, Rectal↗

PubMed2002

Analysis of the immune response and viral evolution during the acute phase of SIV infection.

Development of an effective vaccine against human immunodeficiency virus (HIV) will require knowledge of the immune responses that correlate with protection. During the acute phase of HIV infection the host immune responses appear to control viral replication. It is thought that virus-specific cellular immunity is intimately involved in this viral control. We have developed a model system to measure the entire T cell response and viral evolution in the face of this onslaught in rhesus macaques during the acute phase of infection with molecularly cloned simian immunodeficiency virus (SIV). We used intracellular cytokine staining (ICS) of peripheral blood mononuclear cells (PBMCs) from animals during the acute phase of viral infection stimulated with peptides spanning the entire protein sequence of SIV to determine which peptides were recognized by CD8 and CD4 positive T cells. Furthermore, we sequenced the entire virus during the acute phase of infection. This approach has proved highly effective for measuring acute phase T cell responses and viral evolution in SIV-infected rhesus macaques and might facilitate the definition of cellular immune responses in HIV-infected humans during the acute phase.

Acute Disease↗

PubMed2002

Acute phase cytotoxic T lymphocyte escape is a hallmark of simian immunodeficiency virus infection.

Cytotoxic T-lymphocyte (CTL) responses peak coincident with the decline in acute HIV viremia. Despite two reports of CTL-resistant HIV variants emerging during acute infection, the contribution of acute CTL escape to HIV pathogenesis remains unclear. Difficulties inherent in studying acute HIV infection can be overcome by modeling virus-host interactions in SIV-infected rhesus macaques. We sequenced 21 complete simian immunodeficiency virus (SIV)mac239 genomes at four weeks post-infection to determine the extent of acute CTL escape. Here we show that viruses from 19 of 21 macaques escaped from CTLs during acute infection and that these escape-selecting CTLs were responsive to lower concentrations of peptide than other SIV-specific CTLs. Interestingly, CTLs that require low peptide concentrations for stimulation (high 'functional avidity') are particularly effective at controlling other viral infections. Our results suggest that acute viral escape from CTLs is a hallmark of SIV infection and that CTLs with high functional avidity can rapidly select for escape variants.

Acute Disease↗

PubMed2002

Immunization of rhesus macaques with a DNA prime/modified vaccinia virus Ankara boost regimen induces broad simian immunodeficiency virus (SIV)-specific T-cell responses and reduces initial viral replication but does not prevent disease progression following challenge with pathogenic SIVmac239.

Producing a prophylactic vaccine for human immunodeficiency virus (HIV) has proven to be a challenge. Most biological isolates of HIV are difficult to neutralize, so that conventional subunit-based antibody-inducing vaccines are unlikely to be very effective. In the rhesus macaque model, some protection was afforded by DNA/recombinant viral vector vaccines. However, these studies used as the challenge virus SHIV-89.6P, which is neutralizable, making it difficult to determine whether the observed protection was due to cellular immunity, humoral immunity, or a combination of both. In this study, we used a DNA prime/modified vaccinia virus Ankara boost regimen to immunize rhesus macaques against nearly all simian immunodeficiency virus (SIV) proteins. These animals were challenged intrarectally with pathogenic molecularly cloned SIVmac239, which is resistant to neutralization. The immunization regimen resulted in the induction of virus-specific CD8(+) and CD4(+) responses in all vaccinees. Although anamnestic neutralizing antibody responses against laboratory-adapted SIVmac251 developed after the challenge, no neutralizing antibodies against SIVmac239 were detectable. Vaccinated animals had significantly reduced peak viremia compared with controls (P < 0.01). However, despite the induction of virus-specific cellular immune responses and reduced peak viral loads, most animals still suffered from gradual CD4 depletion and progressed to disease.

AIDS Vaccines↗

PubMed2002

Dominance of CD8 responses specific for epitopes bound by a single major histocompatibility complex class I molecule during the acute phase of viral infection.

Cytotoxic T-lymphocyte (CTL) responses are thought to control human immunodeficiency virus replication during the acute phase of infection. Understanding the CD8(+) T-cell immune responses early after infection may, therefore, be important to vaccine design. Analyzing these responses in humans is difficult since few patients are diagnosed during early infection. Additionally, patients are infected by a variety of viral subtypes, making it hard to design reagents to measure their acute-phase immune responses. Given the complexities in evaluating acute-phase CD8(+) responses in humans, we analyzed these important immune responses in rhesus macaques expressing a common rhesus macaque major histocompatibility complex class I molecule (Mamu-A*01) for which we had developed a variety of immunological assays. We infected eight Mamu-A*01-positive macaques and five Mamu-A*01-negative macaques with the molecularly cloned virus SIV(mac)239 and determined all of the simian immunodeficiency virus-specific CD8(+) T-cell responses against overlapping peptides spanning the entire virus. We also monitored the evolution of particular CD8(+) T-cell responses by tetramer staining of peripheral lymphocytes as well as lymph node cells in situ. In this first analysis of the entire CD8(+) immune response to autologous virus we show that between 2 and 12 responses are detected during the acute phase in each animal. CTL against the early proteins (Tat, Rev, and Nef) and against regulatory proteins Vif and Vpr dominated the acute phase. Interestingly, CD8(+) responses against Mamu-A*01-restricted epitopes Tat(28-35)SL8 and Gag(181-189)CM9 were immunodominant in the acute phase. After the acute phase, however, this pattern of reactivity changed, and the Mamu-A*01-restricted response against the Gag(181-189)CM9 epitope became dominant. In most of the Mamu-A*01-positive macaques tested, CTL responses against epitopes bound by Mamu-A*01 dominated the CD8(+) cellular immune response.

Acute Disease↗

PubMed2002

Effects of cytotoxic T lymphocytes (CTL) directed against a single simian immunodeficiency virus (SIV) Gag CTL epitope on the course of SIVmac239 infection.

Vaccine-induced cytotoxic T lymphocytes (CTL) have been implicated in the control of virus replication in simian immunodeficiency virus (SIV)-challenged and simian-human immunodeficiency virus-challenged macaques. Therefore, we wanted to test the impact that vaccine-induced CTL responses against an immunodominant Gag epitope might have in the absence of other immune responses. By themselves, these strong CTL responses failed to control SIVmac239 replication.

Animals↗

PubMed2002

Escape in one of two cytotoxic T-lymphocyte epitopes bound by a high-frequency major histocompatibility complex class I molecule, Mamu-A*02: a paradigm for virus evolution and persistence?

It is now accepted that an effective vaccine against AIDS must include effective cytotoxic-T-lymphocyte (CTL) responses. The simian immunodeficiency virus (SIV)-infected rhesus macaque is the best available animal model for AIDS, but analysis of macaque CTL responses has hitherto focused mainly on epitopes bound by a single major histocompatibility complex (MHC) class I molecule, Mamu-A*01. The availability of Mamu-A*01-positive macaques for vaccine studies is therefore severely limited. Furthermore, it is becoming clear that different CTL responses are able to control immunodeficiency virus replication with varying success, making it a priority to identify and analyze CTL responses restricted by common MHC class I molecules other than Mamu-A*01. Here we describe two novel epitopes derived from SIV, one from Gag (Gag(71-79) GY9), and one from the Nef protein (Nef(159-167) YY9). Both epitopes are bound by the common macaque MHC class I molecule, Mamu-A*02. The sequences of these two eptiopes are consistent with the molecule's peptide-binding motif, which we have defined by elution of natural ligands from Mamu-A*02. Strikingly, we found evidence for the selection of escape variant viruses by CTL specific for Nef(159-167) YY9 in 6 of 6 Mamu-A*02-positive animals. In contrast, viral sequences encoding the Gag(71-79) GY9 epitope remained intact in each animal. This situation is reminiscent of Mamu-A*01-restricted CTL that recognize Tat(28-35) SL8, which reproducibly selects for escape variants during acute infection, and Gag(181-189) CM9, which does not. Differential selection by CTL may therefore be a paradigm of immunodeficiency virus infection.

Amino Acid Sequence↗