Specificity of CD8+ T-Cell Responses Following Vaccination with Conserved Regions of HIV-1 in Nairobi, Kenya.

Sub-Saharan Africa carries the biggest burden of the human immunodeficiency virus type 1 (HIV-1)/AIDS epidemic and is in an urgent need of an effective vaccine. CD8+ T cells are an important component of the host immune response to HIV-1 and may need to be harnessed if a vaccine is to be effective. CD8+ T cells recognize human leukocyte antigen (HLA)-associated viral epitopes and the HLA alleles vary significantly among different ethnic groups. It follows that definition of HIV-1-derived peptides recognized by CD8+ T cells in the geographically relevant regions will critically guide vaccine development. Here, we study fine details of CD8+ T-cell responses elicited in HIV-1/2-uninfected individuals in Nairobi, Kenya, who received a candidate vaccine delivering conserved regions of HIV-1 proteins called HIVconsv. Using 10-day cell lines established by in vitro peptide restimulation of cryopreserved PBMC and stably HLA-transfected 721.221/C1R cell lines, we confirm experimentally many already defined epitopes, for a number of epitopes we define the restricting HLA molecule(s) and describe four novel HLA-epitope pairs. We also identify specific dominance patterns, a promiscuous T-cell epitope and a rescue of suboptimal T-cell epitope induction in vivo by its functional variant, which all together inform vaccine design.

Randomized phase I trial HIV-CORE 003: Depletion of serum amyloid P component and immunogenicity of DNA vaccination against HIV-1.

BACKGROUND: The failure of DNA vaccination in humans, in contrast to its efficacy in some species, is unexplained. Observational and interventional experimental evidence suggests that DNA immunogenicity may be prevented by binding of human serum amyloid P component (SAP). SAP is the single normal DNA binding protein in human plasma. The drug (R)-1-[6-[(R)-2-carboxypyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid (CPHPC, miridesap), developed for treatment of systemic amyloidosis and Alzheimer's disease, depletes circulating SAP by 95-99%. The proof-of-concept HIV-CORE 003 clinical trial tested whether SAP depletion by CPHPC would enhance the immune response in human volunteers to DNA vaccination delivering the HIVconsv immunogen derived from conserved sub-protein regions of HIV-1. METHODS: Human volunteers received 3 intramuscular immunizations with an experimental DNA vaccine (DDD) expressing HIV-1-derived immunogen HIVconsv, with or without prior depletion of SAP by CPHPC. All subjects were subsequently boosted by simian (chimpanzee) adenovirus (C)- and poxvirus MVA (M)-vectored vaccines delivering the same immunogen. After administration of each vaccine modality, the peak total magnitudes, kinetics, functionality and memory subsets of the T-cell responses to HIVconsv were thoroughly characterized. RESULTS: No differences were observed between the CPHPC treated and control groups in any of the multiple quantitative and qualitative parameters of the T-cell responses to HIVconsv, except that after SAP depletion, there was a statistically significantly greater breadth of T-cell specificities, that is the number of recognized epitopes, following the DDDC vaccination. CONCLUSIONS: The protocol used here for SAP depletion by CPHPC prior to DNA vaccination produced only a very modest suggestion of enhanced immunogenicity. Further studies will be required to determine whether SAP depletion might have a practical value in DNA vaccination for other plasmid backbones and/or immunogens. TRIAL REGISTRATION: Clinicaltrials.gov NCT02425241.

Long-term follow up of human T-cell responses to conserved HIV-1 regions elicited by DNA/simian adenovirus/MVA vaccine regimens.

BACKGROUND: Durability of vaccine-elicited immune responses is one of the key determinants for vaccine success. Our aim is to develop a vaccination strategy against the human immunodeficiency virus type 1 (HIV-1), which induces protective and durable CD8+ T-cell responses. The central theorem of our approach is to focus T cells on highly conserved regions of the HIV-1 proteome and this is achieved through the use of the first-generation conserved vaccine immunogen HIVconsv. This immunogen vectored by plasmid DNA, simian adenovirus and poxvirus MVA was tested in healthy, HIV-1-negative adults in UK and induced high magnitudes of HIVconsv-specific plurifunctional CD8+ T cells capable of in vitro HIV-1 inhibition. Here, we assessed the durability of these responses. METHODS: Vaccine recipients in trial HIV-CORE 002 were invited to provide a blood sample at 1 and 2 years after vaccination. Their PBMCs were tested in IFN-γ ELISPOT, 25-analyte Luminex, CFSE proliferation and intracellular cytokine staining assays, the last enhanced by HLA-peptide dextramer analysis. RESULTS: 12/12 (1 year) and 8/8 (2 years) returning subjects had median (range) of 990 (150-2495) and 763 (70-1745) IFN-γ SFU/106 PBMC specific for HIVconsv, respectively, and recognized 5 (1-6) out of 6 peptide pools at 2 years. Over one-half of the HIVconsv-specific cells expressed at least 3 functions IFN-γ, TNF-α and CD107a, and were capable of proliferation. Among dextramer-reactive cells, naïve, transitional, effector and terminally differentiated memory subsets were similarly represented. CONCLUSIONS: First generation HIVconsv vaccine induced human T cells, which were plurifunctional and persisted for at least 2 years. TRIAL REGISTRATION: ClinicalTrials.gov NCT01151319.

Broad HIV-1 inhibition in vitro by vaccine-elicited CD8(+) T cells in African adults.

We are developing a pan-clade HIV-1 T-cell vaccine HIVconsv, which could complement Env vaccines for prophylaxis and be a key to HIV cure. Our strategy focuses vaccine-elicited effector T-cells on functionally and structurally conserved regions (not full-length proteins and not only epitopes) of the HIV-1 proteome, which are common to most global variants and which, if mutated, cause a replicative fitness loss. Our first clinical trial in low risk HIV-1-negative adults in Oxford demonstrated the principle that naturally mostly subdominant epitopes, when taken out of the context of full-length proteins/virus and delivered by potent regimens involving combinations of simian adenovirus and poxvirus modified vaccinia virus Ankara, can induce robust CD8(+) T cells of broad specificities and functions capable of inhibiting in vitro HIV-1 replication. Here and for the first time, we tested this strategy in low risk HIV-1-negative adults in Africa. We showed that the vaccines were well tolerated and induced high frequencies of broadly HIVconsv-specific plurifunctional T cells, which inhibited in vitro viruses from four major clades A, B, C, and D. Because sub-Saharan Africa is globally the region most affected by HIV-1/AIDS, trial HIV-CORE 004 represents an important stage in the path toward efficacy evaluation of this highly rational and promising vaccine strategy.

Control of HIV-1 replication in vitro by vaccine-induced human CD8(+) T cells through conserved subdominant Pol epitopes.

OBJECTIVE: The specificity of CD8(+) T cells is critical for early control of founder/transmitted and reactivated HIV-1. To tackle HIV-1 variability and escape, we designed vaccine immunogen HIVconsv assembled from 14 highly conserved regions of mainly Gag and Pol proteins. When administered to HIV-1-negative human volunteers in trial HIV-CORE 002, HIVconsv vaccines elicited CD8(+) effector T cells which inhibited replication of up to 8 HIV-1 isolates in autologous CD4(+) cells. This inhibition correlated with interferon-γ production in response to Gag and Pol peptide pools, but direct evidence of the inhibitory specificity was missing. Here, we aimed to define through recognition of which epitopes these effectors inhibit HIV-1 replication. DESIGN: CD8(+) T-cells from the 3 broadest HIV-1 inhibitors out of 23 vaccine recipients were expanded in culture by Gag or Pol peptide restimulation and tested in viral inhibition assay (VIA) using HIV-1 clade B and A isolates. METHODS: Frozen PBMCs were expanded first using peptide pools from Gag or Pol conserved regions and tested on HIV-1-infected cells in VIA or by individual peptides for their effector functions. Single peptide specificities responsible for inhibition of HIV-1 replication were then confirmed by single-peptide expanded effectors tested on HIV-1-infected cells. RESULTS: We formally demonstrated that the vaccine-elicited inhibitory human CD8(+) T cells recognized conserved epitopes of both Pol and Gag proteins. We defined 7 minimum epitopes, of which 3 were novel, presumably naturally subdominant. The effectors were oligofunctional producing several cytokines and chemokines and killing peptide-pulsed target cells. CONCLUSIONS: These results implicate the use of functionally conserved regions of Pol in addition to the widely used Gag for T-cell vaccine design. Proportion of volunteers developing these effectors and their frequency in circulating PBMC are separate issues, which can be addressed, if needed, by more efficient vector and regimen delivery of conserved immunogens.

Humoral responses to HIVconsv induced by heterologous vaccine modalities in rhesus macaques.

Vaccines delivering T cell immunogen HIVconsv vectored by plasmid DNA, non-replicating simian adenovirus and non-replicating modified vaccinia virus Ankara (MVA) are under clinical evaluation in phase I/IIa trials in UK, Europe, and Africa. While these vaccines aim to induce effector T cell responses specific for HIV-1, we here characterized the humoral responses induced by HIVconsv administration to macaques using six different vaccine modalities: plasmid DNA, human adenovirus serotype 5, simian adenovirus serotype 63, MVA, Semliki Forest virus replicons, and adjuvanted overlapping synthetic long peptides (SLP). We found that only the SLP formulation, but none of the genetic vaccine platforms induced antibodies recognizing linear HIVconsv epitopes, median 32/46 SLP.HIVconsv peptides. These antibodies bound to 15-mer and SLP peptides, recombinant gp120 and trimeric gp140 of HIV-1 Bal, YU2, JRFL, and UG037, but failed to react with HIV-1 Bal and IIIB virions and HIV-1 Bal- and IIIB-infected human cells, and consequently failed to induce neutralizing antibodies. The HIVconsv immunogen contains conserved regions derived from Gag, Pol, Vif, and Env proteins of HIV-1, and antibodies induced by the SLP.HIVconsv vaccination resulted in positive signals in routine HIV-1 tests. Thus, only HIVconsv delivered by SLP resulted in seroconversion, an observation that provides important guidance for recruiting volunteers into future clinical trials. Furthermore, our data confirms that vaccine delivery by SLP induces humoral as well as cellular immune responses and could be considered for inclusion in future vaccine regimens where this is required.

The biology of micrometastases from uveal melanoma.

BACKGROUND: The aim of this study was to investigate the possible causes of tumour latency in uveal melanoma primarily through the analysis of micrometastases in tissue obtained from donors postmortem. Various explanations have been proposed but there is no clear answer from animal studies and few human data. The main hypotheses may be divided into several areas--immunological control of metastatic cells, lack of angiogenesis within micrometastases and reduced cell turnover. METHODS: 196 patients were recruited to the study between 2003 and 2007. Patients were invited to take part and their relatives agreed to postmortem examination of their liver and lungs in the event of their death, including tissue sampling to assess the presence of micrometastases and their biology. Metastatic cells were detected by immunohistochemistry using a pan-melanoma antibody reagent, and by quantitative reverse transcriptase (qRT)-PCR for three melanoma-associated genes (tyrosinase Melan-A, and gp100) and a housekeeping gene (HMBS/PBGD) in samples stored in RNAlater or as formalin-fixed paraffin-embedded tissue. RESULTS: 22 deaths were investigated at autopsy as part of the study. Sixteen patients died with large deposits of metastatic melanoma, while six patients died of other causes. In addition, a liver resection for hepatic adenoma provided further tissue from a case without clinical evidence of metastasis. Metastatic melanoma cells were identified by immunohistochemistry of the liver samples in one case and by qRT-PCR in two further cases without macrometastases. There was no evidence of multicellular micrometastases sufficiently large to require angiogenesis and no associated inflammation was observed. CONCLUSION: The most likely explanation for latency in this setting is the inability of uveal melanoma cells in metastatic sites to grow.

Melanoma vaccines: The problems of local immunosuppression.

The incidence of cutaneous melanoma in Europe is rising, and the disease is incurable once metastases occur. Because melanoma expresses antigens that can be specifically recognized by the immune system, and because this disease occasionally undergoes spontaneous regression mediated by anti-tumor immunity, a number of different melanoma vaccines have been developed and tested clinically. Although most such vaccines show efficacy in vitro and an ability to stimulate anti-melanoma immune responses in blood, they have proved disappointing in clinical practice. It has become increasingly clear that the interaction between melanoma and the immune system is determined locally, within the tumor or draining lymph nodes. It is now clear that melanoma cells have the ability to anergize the immune system by inducing an immunosuppressive microenvironment that may explain the inability of systemic vaccines to alter patient outcomes. This subversion of the immune system involves alteration of dendritic cell (DC) function by tumor-derived cytokines, leading to the generation of suppressive and regulatory T lymphocytes. Successful melanoma vaccination probably requires therapeutic neutralization of the immunosuppressive microenvironment, which will require greater understanding of the molecular mechanisms used by the tumor to promote immunosuppression. Nevertheless, if these problems can be overcome, it seems likely that the efficacy of melanoma vaccines could be greatly enhanced.

Activation of tonsil dendritic cells with immuno-adjuvants.

BACKGROUND: Dendritic cells (DC) play the key role in directing antigen-specific immune responses and manipulating their function may be a useful tool for immunotherapy. The balance between immune stimulation and tolerance is particularly important at mucosal interfaces, where discrimination between dangerous pathogens and innocuous antigens takes place. In humans, although much is known about the responses of monocyte derived DC, relatively little is known about effect of immuno-stimulatory adjuvants on DC found in tonsil. RESULTS: To examine this, tonsil DC were isolated and cultured with potent DC activators; IFNgamma, anti-CD40 antibody, LPS and Poly I:C either singly or in combination. To measure maturation and activation, DC were examined for changes in the expression of HLA-DR, HLA- class I, CD83, CD40, CD80 and CD86 and the release of IL12p70. The DC isolated from tonsil were a mixed population containing both myeloid and plasmacytoid DC, but all showed similar responses. Tonsil DC released IL12p70 upon stimulation with IFNgamma , anti-CD40 antibody, and LPS, but unlike monocyte-derived DC, they did not increase the expression of cell surface activation molecules above those induced by culture alone. Poly I:C, a potent stimulator of laboratory generated DC inhibited the activation of tonsil DC by other adjuvants. CONCLUSION: As the response of this mixed population of DC does not mirror that of DC generated in vitro, this may have implications for other tissue residing DC and might be an important consideration for immunotherapy.

Presence and phenotype of dendritic cells in uveal melanoma.

BACKGROUND: Uveal melanoma arises in an immune-privileged site and can itself add to the immunosuppressive environment. Previous studies on cutaneous melanoma have shown the presence of tolerogenic dendritic cells (DCs), which could play an important role in the progression of the tumour. AIM: To examine the presence and functional status of DCs in a small series of uveal melanomas. METHODS: 10 cases of uveal melanoma were examined for the expression of FXIIIa, CD68, human leucocyte antigen (HLA)-DR, CD40, CD83, transforming growth factor betaR1 and indolamine 2,3 dioxygenase by immunohistochemical analysis on sections embedded in paraffin wax. RESULTS: CD68-positive macrophages were present in all of the tumours and were evenly distributed throughout. DCs expressing FXIIIa-positive were seen in 7 cases, and were often found concentrated in foci within the tumour mass. These cells were dendritic and expressed high levels of HLA-DR. The DCs did not express the maturation markers CD83 or CD40. In one case, concentration of DCs around the area of tumour necrosis was observed, and some of these cells expressed CD83. CONCLUSION: Numerous tolerising antigen-presenting cells may play a role in melanoma-related immunosuppression in the eye, although activation of DCs may be associated with tumour necrosis.

Post-menopausal bleeding: a rare presentation of metastatic uveal melanoma.

Uveal melanoma differs from cutaneous melanoma in many ways, including its pattern of metastasis, and exhibits latency with clinical evidence of metastasis sometimes appearing many years after primary diagnosis. Most patients develop metastasis within the liver, but some may present with metastasis to other sites. We report a case of uveal melanoma that presented with post-menopausal bleeding due to metastasis. Further investigation revealed widespread metastatic disease and the patient was not fit for chemotherapy. She died two months after presentation: autopsy revealed metastases in many sites, including the uterus, right ovarian fibroma, kidney, mesentery, liver, lung, thyroid, bone marrow and skin. The immediate cause of death was cardiac tamponade due to a malignant effusion secondary to cardiac metastasis. This case illustrates the widespread metastatic potential of uveal melanoma and highlights the potential for unusual presentation of metastatic disease from this eye tumor.

Presence and maturity of dendritic cells in melanoma lymph node metastases.

Immune avoidance mechanisms play a key role in the successful dissemination of melanoma. One mechanism whereby this could be achieved is by interfering with dendritic cell (DC) presentation of tumour-associated antigens to naïve T cells. In particular, immature DCs characterized by the absence of accessory molecules are known to be immunosuppressive and to be involved in the induction of tolerance. The present study has investigated the presence and activation status of DCs within melanoma metastases in the regional lymph nodes. Using image analysis techniques, the expression of Factor XIIIa (FXIIIa), CD40, CD83 and HLA-DR and the morphological features of DCs were examined in paraffin sections from 26 lymph nodes containing melanoma metastases. DCs expressing FXIIIa were found in 70% of the lymph nodes. The number of DCs identified was generally small but there were more concentrated areas of DCs designated as hotspots. In these areas of high FXIIIa staining, the percentage area occupied by DCs varied between 0.1% and 10%. The majority of FXIIIa-positive cells did not express the DC maturation markers CD83 or CD40 and morphologically were rounded with few dendrites, indicating that they were immature. The cells did, however, express high levels of HLA-DR, suggesting that they have the ability to present antigen but lack the accessory molecules required to initiate an immune response. Immature DCs, characterized by phenotypic and morphological features, are therefore present within the tumour deposits in lymph nodes infiltrated by melanoma and may specifically modulate the anti-melanoma immune response.

Virus-specific CTL responses induced by an H-2K(d)-restricted, motif-negative 15-mer peptide from the fusion protein of respiratory syncytial virus.

We describe 15-mer peptide P8:F92-106 from the F protein of respiratory syncytial virus (RSV) that can act as an MHC class I-restricted (H-2K(d)) epitope for RSV-specific CD8(+) CTL. This peptide is interesting because not only is it the first murine CTL epitope to be identified in the F protein but also because it does not contain a known allele-specific motif, as all 15 amino acids appear to be required for effective presentation to CTL. In in vitro MHC class I refolding experiments, peptide P8:F92-106 induced complex formation with H-2K(d) heavy chains and beta2-microglobulin. Immunization of BALB/c mice with P8:F92-106 resulted in the induction of peptide and RSV-specific CTL responses as well as peptide-specific proliferative responses. Following intranasal challenge with RSV, P8:F92-106-immunized mice showed a significant reduction in viral load in the lungs compared to that seen in unimmunized mice. Furthermore, passive transfer of purified CD8(+) lymphocytes into BALB/c scid mice prior to challenge with RSV also resulted in a reduction in the virus load in lungs of challenged mice. These results indicate the potential of synthetic peptide epitopes for the induction of protective immune responses against RSV infection.