Nicola J. Borthwick

Lymphocyte activation in HIV-1 infection. II: Functional defects of CD28-T cells

Objectives and design:The expression of the accessory molecule CD28 was compared in various populations of T and natural killer (NK) cells from HIV-1-negative and HIV-1-positive individuals and correlated with activation using mitogens in vitro. Methods:Multiparameter flow cytometric analysis using combinations of CD3 CD28 and other markers was performed together with absolute cell counting in peripheral blood. Blast transformation and proliferative responses were also quantitated using the Cytoronabsolute after stimulation with phytohaemagglutinin (PHA) and anti-CD3. CD28− cells were also purified to confirm the observations. Results:In HIV-1-negative individuals >90% of CD3+ T cells were CD28+ and responded to stimulation, while CD3− CD16+ CD57+ NK-like cells were CD28-and failed to respond. In HIV-1-positive individuals the expression of CD28 was greatly reduced and the proportion of CD3+CD28− T cells expanded. CD8 lymphocytosis was caused entirely by the accumulation of CD28− T cells and many of these expressed activation markers human lymphocyte antigen-DR, CD38 and CD45RO on their membrane and molecules such as TIA-1 and perform, associated with cytolytic function, in their cytoplasm. The strong positive correlation (r = 0.66) between the lack of CD28 expression and the poor proliferation from HIV-1-positive individuals was confirmed by demonstrating that only CD28+ cells transformed into lymphoblasts and proliferated. Although the CD28− including CD3+ T cells transiently expressed CD25 (interleukin-2Rct), they did not undergo blastogenesis or activation measured by bromodeoxyuridine uptake and died after 3–4 days in culture. These observations were confirmed in costimulation experiments with anti-CD2 and anti-CD28. Conclusion:In HIV-1 infection activated CD3+CD28− T cells accumulate but are unresponsive to mitogens and anti-CD28. These cells appear to represent terminally differentiated effector cells which fail to respond to further stimuli because of the absence of a CD28 second signal.

Vaccine-elicited Human T Cells Recognizing Conserved Protein Regions Inhibit HIV-1

Virus diversity and escape from immune responses are the biggest challenges to the development of an effective vaccine against HIV-1. We hypothesized that T-cell vaccines targeting the most conserved regions of the HIV-1 proteome, which are common to most variants and bear fitness costs when mutated, will generate effectors that efficiently recognize and kill virus-infected cells early enough after transmission to potentially impact on HIV-1 replication and will do so more efficiently than whole protein-based T-cell vaccines. Here, we describe the first-ever administration of conserved immunogen vaccines vectored using prime-boost regimens of DNA, simian adenovirus and modified vaccinia virus Ankara to uninfected UK volunteers. The vaccine induced high levels of effector T cells that recognized virus-infected autologous CD4(+) cells and inhibited HIV-1 replication by up to 5.79 log10. The virus inhibition was mediated by both Gag- and Pol- specific effector CD8(+) T cells targeting epitopes that are typically subdominant in natural infection. These results provide proof of concept for using a vaccine to target T cells at conserved epitopes, showing that these T cells can control HIV-1 replication in vitro.

Long peptides induce polyfunctional T cells against conserved regions of HIV‐1 with superior breadth to single‐gene vaccines in macaques

A novel T‐cell vaccine strategy designed to deal with the enormity of HIV‐1 variation is described and tested for the first time in macaques to inform and complement approaching clinical trials. T‐cell immunogen HIVconsv, which directs vaccine‐induced responses to the most conserved regions of the HIV‐1, proteome and thus both targets diverse clades in the population and reduces the chance of escape in infected individuals, was delivered using six different vaccine modalities: plasmid DNA (D), attenuated human (A) and chimpanzee (C) adenoviruses, modified vaccinia virus Ankara (M), synthetic long peptides, and Semliki Forest virus replicons. We confirmed that the initial DDDAM regimen, which mimics one of the clinical schedules (DDDCM), is highly immunogenic in macaques. Furthermore, adjuvanted synthetic long peptides divided into sub‐pools and delivered into anatomically separate sites induced T‐cell responses that were markedly broader than those elicited by traditional single‐open‐reading‐frame genetic vaccines and increased by 30% the overall response magnitude compared with DDDAM. Thus, by improving both the HIV‐1‐derived immunogen and vector regimen/delivery, this approach could induce stronger, broader, and theoretically more protective T‐cell responses than vaccines previously used in humans.

Prime-boost regimens with adjuvanted synthetic long peptides elicit T cells and antibodies to conserved regions of HIV-1 in macaques.

Objectives:Administration of synthetic long peptides (SLPs) derived from human papillomavirus to cervical cancer patients resulted in clinical benefit correlated with expansions of tumour-specific T cells. Because vaginal mucosa is an important port of entry for HIV-1, we have explored SLP for HIV-1 vaccination. Using immunogen HIVconsv derived from the conserved regions of HIV-1, we previously showed in rhesus macaques that SLP.HIVconsv delivered as a boost increased the breath of T-cell specificities elicited by single-gene vaccines. Here, we compared and characterized the use of electroporated pSG2.HIVconsv DNA (D) and imiquimod/montanide-adjuvanted SLP.HIVconsv (S) as priming vaccines for boosting with attenuated chimpanzee adenovirus ChAdV63.HIVconsv (C) and modified vaccinia virus Ankara MVA.HIVconsv (M). Design:Prime-boost regimens of DDDCMS, DSSCMS and SSSCMS in rhesus macaques. Methods:Animals’ blood was analysed regularly throughout the vaccination for HIV-1-specific T-cell and antibody responses. Results:We found that electroporation spares DNA dose, both SLP.HIVconsv and pSG2.HIVconsv DNA primed weakly HIVconsv-specific T cells, regimen DDDCM induced the highest frequencies of oligofunctional, proliferating CD4+ and CD8+ T cells, and a subsequent SLP.HIVconsv boost expanded primarily CD4+ cells. DSS was the most efficient regimen inducing antibodies binding to regions of trimeric HIV-1 Env, which are highly conserved among the four major global clades, although no unequivocal neutralizing activity was detected. Conclusion:The present results encourage evaluation of the SLP.HIVconsv vaccine modality in human volunteers along the currently trialled pSG2.HIVconsv DNA, ChAdV63.HIVconsv and MVA.HIVconsv vaccines. These results are discussed in the context of the RV144 trial outcome.

Safety and immunogenicity of novel recombinant BCG and modified vaccinia virus Ankara vaccines in neonate rhesus macaques.

ABSTRACT Although major inroads into making antiretroviral therapy available in resource-poor countries have been made, there is an urgent need for an effective vaccine administered shortly after birth, which would protect infants from acquiring human immunodeficiency virus type 1 (HIV-1) through breast-feeding. Bacillus Calmette-Guérin (BCG) is given to most infants at birth, and its recombinant form could be used to prime HIV-1-specific responses for a later boost by heterologous vectors delivering the same HIV-1-derived immunogen. Here, two groups of neonate Indian rhesus macaques were immunized with either novel candidate vaccine BCG.HIVA401 or its parental strain AERAS-401, followed by two doses of recombinant modified vaccinia virus Ankara MVA.HIVA. The HIVA immunogen is derived from African clade A HIV-1. All vaccines were safe, giving local reactions consistent with the expected response at the injection site. No systemic adverse events or gross abnormality was seen at necropsy. Both AERAS-401 and BCG.HIVA401 induced high frequencies of BCG-specific IFN-γ-secreting lymphocytes that declined over 23 weeks, but the latter failed to induce detectable HIV-1-specific IFN-γ responses. MVA.HIVA elicited HIV-1-specific IFN-γ responses in all eight animals, but, except for one animal, these responses were weak. The HIV-1-specific responses induced in infants were lower compared to historic data generated by the two HIVA vaccines in adult animals but similar to other recombinant poxviruses tested in this model. This is the first time these vaccines were tested in newborn monkeys. These results inform further infant vaccine development and provide comparative data for two human infant vaccine trials of MVA.HIVA.

Activation-associated necrosis in human immunodeficiency virus infection.

Mitogenic stimulation of lymphocytes from persons infected with human immunodeficiency virus (HIV) resulted in massive cell death. In addition to early apoptosis, a second wave of cell death occurred 48-72 h after stimulation. At that time, the cells were enlarged, leaked content, and had plasma membrane damage-all indicative of necrosis. Furthermore, DNA fragmentation as determined by TUNEL assay was virtually absent. This activation-associated necrosis could not be prevented by interfering with CD95/CD95-ligand interactions or by blocking caspase activity and was unaffected by neutralizing antibodies to tumor necrosis factor-alpha or interferon-gamma. Necrosis was also induced by activation of normal lymphocytes in the presence of ribavirin, which inhibits the de novo pathway of nucleotide synthesis. Lymphocytes from HIV-infected persons are defective in their ability to synthesize nucleotides via this pathway, indicating one possible mechanism for the activation-associated necrosis seen in HIV infection.

Novel Recombinant Mycobacterium bovis BCG, Ovine Atadenovirus, and Modified Vaccinia Virus Ankara Vaccines Combine To Induce Robust Human Immunodeficiency Virus-Specific CD4 and CD8 T-Cell Responses in Rhesus Macaques

ABSTRACT Mycobacterium bovis bacillus Calmette-Guérin (BCG), which elicits a degree of protective immunity against tuberculosis, is the most widely used vaccine in the world. Due to its persistence and immunogenicity, BCG has been proposed as a vector for vaccines against other infections, including HIV-1. BCG has a very good safety record, although it can cause disseminated disease in immunocompromised individuals. Here, we constructed a recombinant BCG vector expressing HIV-1 clade A-derived immunogen HIVA using the recently described safer and more immunogenic BCG strain AERAS-401 as the parental mycobacterium. Using routine ex vivo T-cell assays, BCG.HIVA401 as a stand-alone vaccine induced undetectable and weak CD8 T-cell responses in BALB/c mice and rhesus macaques, respectively. However, when BCG.HIVA401 was used as a priming component in heterologous vaccination regimens together with recombinant modified vaccinia virus Ankara-vectored MVA.HIVA and ovine atadenovirus-vectored OAdV.HIVA vaccines, robust HIV-1-specific T-cell responses were elicited. These high-frequency T-cell responses were broadly directed and capable of proliferation in response to recall antigen. Furthermore, multiple antigen-specific T-cell clonotypes were efficiently recruited into the memory pool. These desirable features are thought to be associated with good control of HIV-1 infection. In addition, strong and persistent T-cell responses specific for the BCG-derived purified protein derivative (PPD) antigen were induced. This work is the first demonstration of immunogenicity for two novel vaccine vectors and the corresponding candidate HIV-1 vaccines BCG.HIVA401 and OAdV.HIVA in nonhuman primates. These results strongly support their further exploration.

Novel mechanism for the impairment of cell proliferation in HIV-1 infection

The synthesis of ribonucleotides is essential to cell proliferation. Defects in the relevant metabolic pathways have been demonstrated in stimulated T cells from AIDS patients and are associated with lymphocyte necrotic death. Here, Margarita Bofill and colleagues discuss the possibility that an impaired ribonucleotide metabolism might be common to all rapidly dividing cells and thus contribute to other recognized symptoms of HIV-1 infection.

Flow cytometric analysis of the stimulatory response of T cell subsets from normal and HIV-1+ individuals to various mitogenic stimuli in vitro

A novel technique is described which allows the study of the responses of T cell subpopulations stimulated in bulk cultures without interfering with cell‐cell interactions. The number and phenotype of lymphoblasts developing following stimulation with phytohaemagglutinin (PHA), anti‐CD3, staphylococcal protein A (SPA) and pokeweed mitogen (PWM) was determined in HIV‐P and HIV‐1+ patients using a new five‐parameter flow cytometric method. We found that normal T ceils responded faster to PHA than lo any of the other mitogens tested. The peak of the PHA response occurred on day 3. followed by anti‐CD3 and SPA on day 4 and PWM mitogen on day 5. Although PHA and anti‐CD3 stimulated up to 95% and 80% of lymphocytes, respectively, SPA and PWM stimulated only 40% and 30% of cells, respectively. A defective T cell response was observed in lymphocytes cultured from asymptomatic HlV‐1+ patients compared with negative controls. This loss of response was related to a selective mortality of T cells following mitogenic stimulation, referred to as activation‐associated lymphocyte death (AALD). The results showed that stronger mitogens (PHA and anti‐CD3) induced AALD in a larger proportion (50‐60%) of T cells than weaker mitogens such as SPA and PWM (50‐40%). and that AALD affected different lymphocyte subsets to different extents. AALD occurred more frequently in total CD4+ and CD45RO+ T ceils compared with CD4+ and CD45RA+ T cells, but memory CD4+ T cells were the population most severely affected in samples from HIV‐I+ donors.

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.