Prospects for vaccines to protect against AIDS, tuberculosis, and malaria

Given the scope of the worldwide health problems caused by the acquired immunodeficiency syndrome, tuberculosis, and malaria, it is imperative that vaccines be developed to prevent these infections. Recent advances in the understanding of these diseases suggest that T-lymphocyte-mediated immunity is important in containing these infections. The application of novel vaccine technologies for eliciting this type of immunity promises to provide successful vaccines for controlling the spread of these deadly infections.     

Equivalent induction of telomerase-specific cytotoxic T lymphocytes from tumor-bearing patients and healthy individuals.

Although high frequencies of T lymphocytes specific for certain tumor-associated antigens have been detected in some cancer patients, increasing evidence suggests that these T cells may be functionally defective in vivo and fail to induce meaningful clinical responses. One strategy to overcome this limitation is to target novel antigens that are ignored during the natural antitumor immune response but are nevertheless capable of triggering effector T-cell responses against tumors after optimal presentation by antigen-presenting cells. Here, we show that the telomerase catalytic subunit (hTERT)-a nearly universal tumor antigen identified by epitope deduction rather than from patient immune responses-is immunologically ignored by patients despite progressive tumor burden. Nevertheless, HLA-A2-restricted CTLs against hTERT are equivalently induced ex vivo from patients and healthy individuals and efficiently kill human tumor cell lines and primary tumors. Thus, telomerase-specific T cells from cancer patients are spared functional inactivation because of immunological ignorance. These findings support clinical efforts to target the hTERT as a tumor antigen with broad therapeutic potential.     

Low-dose rectal inoculation of rhesus macaques by SIVsmE660 or SIVmac251 recapitulates human mucosal infection by HIV-1

We recently developed a novel strategy to identify transmitted HIV-1 genomes in acutely infected humans using single-genome amplification and a model of random virus evolution. Here, we used this approach to determine the molecular features of simian immunodeficiency virus (SIV) transmission in 18 experimentally infected Indian rhesus macaques. Animals were inoculated intrarectally (i.r.) or intravenously (i.v.) with stocks of SIVmac251 or SIVsmE660 that exhibited sequence diversity typical of early-chronic HIV-1 infection. 987 full-length SIV env sequences (median of 48 per animal) were determined from plasma virion RNA 1–5 wk after infection. i.r. inoculation was followed by productive infection by one or a few viruses (median 1; range 1–5) that diversified randomly with near starlike phylogeny and a Poisson distribution of mutations. Consensus viral sequences from ramp-up and peak viremia were identical to viruses found in the inocula or differed from them by only one or a few nucleotides, providing direct evidence that early plasma viral sequences coalesce to transmitted/founder viruses. i.v. infection was >2,000-fold more efficient than i.r. infection, and viruses transmitted by either route represented the full genetic spectra of the inocula. These findings identify key similarities in mucosal transmission and early diversification between SIV and HIV-1, and thus validate the SIV–macaque mucosal infection model for HIV-1 vaccine and microbicide research.An effective HIV-1 vaccine, microbicide, or other pre- or post-exposure prophylactic must interdict virus at or near the moment of mucosal transmission or in the early period preceding the establishment of viral latency and disseminated infection (1–4). In humans, it has been difficult to study these earliest viral host events in vivo (2, 5–13), and in tissue explant cultures or in Indian rhesus macaques the HIV-1 or simian immunodeficiency virus (SIV) inocula have typically been high to achieve uniform infection of controls or to visualize infection events in situ (14–20), thus prompting concerns about the physiological relevance of the model systems (21–24). Further complicating the analysis of early infection events in vivo is the viral “eclipse” period during which virus replicates in mucosal and locoregional lymphoreticular tissues but is not yet detectable in the circulating plasma (25). In SIV-infected macaques, the eclipse period is generally ∼4–7 d in duration, and in HIV-1–infected humans, it is ∼7–21 d (5, 18, 25–27).Previously, we observed that in the early stages of HIV-1 infection preceding antibody seroconversion (eclipse phase and Fiebig stages I and II [25]), virus diversification follows a pattern of random evolution with an almost starlike phylogeny and a Poisson distribution of nucleotide substitutions (5). We thus hypothesized that the genetic identity of transmitted or early founder viruses could be inferred unambiguously by phylogenetic analysis of discrete low-diversity viral lineages that emanate from them. This hypothesis was supported by an analysis of 3,449 full-length env genes from 102 human subjects with acute HIV-1 subtype B infection, where we found that (a) acute viral sequences sampled before the development of measurable adaptive immune responses conformed to a pattern of random virus evolution; (b) viral sequence diversity resulted in model estimates of time to a most recent common ancestor (MRCA) that was consistent with clinical histories and Fiebig stage classifications; and (c) in most subjects (78 of 102) there was evidence of productive infection by only a single virus, whereas in 24 other subjects infection resulted from transmission of at least 2 to 5 viruses, each recognizable as a discrete virus lineage (5). These findings have since been corroborated in seven additional patient cohorts infected by HIV-1 subtypes A, B, C, or D (28–35). A key innovation common to these studies was the use of singe-genome amplification (SGA) of plasma viral RNA, followed by direct amplicon sequencing to characterize the virus quasispecies (5, 35–39). This method provides proportional representation of plasma viral RNA (vRNA) and precludes Taq polymerase-induced template switching (recombination), Taq polymerase-associated nucleotide substitutions in finished sequences, template resampling, and cloning bias (5, 35, 37, 38, 40).In this study, we sought to directly test our strategy for identifying transmitted/founder viruses in the Indian rhesus macaque SIV infection model where we could define essential experimental parameters, including the route of SIV infection, genetic composition of the inoculum, and the duration between virus inoculation and sampling of plasma vRNA. The primary study objectives were twofold: first, to determine if, as our hypothesis and model predict, plasma SIV sequences sampled at or near peak viremia coalesce to sequences of viruses responsible for transmission and productive clinical infection weeks earlier; and second, to determine how closely a low-dose SIV rectal transmission model in Indian rhesus macaques recapitulates features of human infection by HIV-1, including the extent of the mucosal barrier to virus transmission, the number of transmitted/founder viruses leading to productive infection, and the molecular patterns of early virus diversification.     

Major histocompatibility complex class I molecules of nonhuman primates

The usefulness of nonhuman primates in immunologically relevant research has until now been limited by difficulties in characterizing the major histocompatibility (MHC) gene products of these species. We have now biochemically characterized the MHC-encoded class I molecules from four different species of nonhuman primates using antibodies directed against human MHC class I structures and one-dimensional isoelectric focusing (1-D IEF). We demonstrated the functional relevancy of this technique of MHC typing by generating virus-specific cytotoxic T cells and assaying their cytotoxic activity against a panel of virus-transformed cells that expressed the same or differing class I structures. Only virus-infected cell lines expressing MHC class I antigens identical to those of the cytotoxic T lymphocyte population were lysed. This simple method of MHC class I typing using 1-D IEF will be useful in immunological research involving nonhuman primates and in nonhuman primate colony management.     

An IL-2/Ig fusion protein influences CD4+ T lymphocytes in naive and simian immunodeficiency virus-infected Rhesus monkeys.

The T cell-stimulatory cytokine interleukin 2 (IL-2) is being evaluated as a therapeutic in the clinical settings of HIV infection and cancer. However, the clinical utility of IL-2 may be mitigated by its short in vivo half-life, toxic effects, and high production costs. We show here that an IL-2/Ig fusion protein possesses IL-2 immunostimulatory activity in vitro and a long in vivo half-life. IL-2/Ig treatment of healthy rhesus monkeys induced significant increases in CD4(+) T lymphocyte counts and expression of CD25 by these cells. Short courses of IL-2/Ig treatment of simian immunodeficiency virus (SIV)-infected rhesus monkeys in conjunction with antiretroviral drugs resulted in increased CD25 expression on T lymphocytes, and transient increases in CD4(+) T lymphocyte counts. Plasma viremia did not increase in these treated animals. Treatment of healthy or SIV-infected rhesus monkeys with a plasmid encoding the IL-2/Ig protein did not affect CD4(+) T lymphocytes. These results demonstrate that IL-2/Ig has potential utility as an immunostimulatory therapeutic.     

Animal models for acquired immunodeficiency syndrome

Substantial advances have already been made in the understanding of acquired immunodeficiency syndrome (AIDS). The major issues for AIDS research during the next few years must be practical ones: the development of a safe, effective vaccine for individuals not yet infected with the causative virus and the development of drug therapies for those already infected. Suitable animal models will be needed for studies designed to achieve these goals. Areas of investigation in animal models can be divided into four categories on the basis of increasing direct relevance to AIDS in humans: retroviruses that have no obvious, close relation to human immunodeficiency virus (HIV) but can induce chronic diseases with manifestations that include immunologic abnormalities; ungulate lentiviruses; HIV-related viruses of Old World primates; and HIV infection of chimpanzees. It is hoped that important research developments in experimental models can be quickly extrapolated to human AIDS.     

Delineation of lymphocyte subsets in lymph nodes of nonhuman primates

Monoclonal antibodies which recognize specific surface antigens on human lymphocytes were used with an avidin-biotin immunoperoxidase technique to stain lymph nodes from nonhuman primates. Cells in lymph nodes from three macaque species and two new-world primate species could be stained to define T11, T4, T8, and B1 lymphocyte subsets. This approach will facilitate the study of many important spontaneous and experimentally induced diseases of nonhuman primates.     

Standardized assessment of NAb responses elicited in rhesus monkeys immunized with single- or multi-clade HIV-1 envelope immunogens

The genetic diversity of HIV-1 envelope glycoproteins (Env) remains a major obstacle to the development of an antibody-based AIDS vaccine. The present studies examine the breadth and magnitude of neutralizing antibody (NAb) responses in rhesus monkeys after immunization with DNA prime-recombinant adenovirus (rAd) boost vaccines encoding either single or multiple genetically distant Env immunogens, and subsequently challenged with a pathogenic simian-human immunodeficiency virus (SHIV-89.6P). Using a standardized multi-tier panel of reference Env pseudoviruses for NAb assessment, we show that monkeys immunized with a mixture of Env immunogens (clades A, B, and C) exhibited a greater breadth of NAb activity against neutralization-sensitive Tier 1 viruses following both vaccination and challenge compared to monkeys immunized with a single Env immunogen (clade B or C). However, all groups of Env-vaccinated monkeys demonstrated only limited neutralizing activity against Tier 2 pseudoviruses, which are more characteristic of the neutralization sensitivity of circulating HIV-1. Notably, the development of a post-challenge NAb response against SHIV-89.6P was similar in monkeys receiving either clade B, clade C, or clade A+B+C Env immunogens, suggesting cross-clade priming of NAb responses. In addition, vaccines encoding Env immunogens heterologous to SHIV-89.6P primed for a rapid anamnestic NAb response following infection compared to vaccines lacking an Env component. These results show that DNA/rAd immunization with multiple diverse Env immunogens is a viable approach for enhancing the breadth of NAb responses against HIV-1, and suggest that Env immunogens can prime for anamnestic NAb responses against a heterologous challenge virus.     

Beta-amino ester polymers facilitate in vivo DNA transfection and adjuvant plasmid DNA immunization.

Increased in vivo expression of intramuscularly delivered plasmid DNA will be essential for clinical success in gene therapy and plasmid DNA vaccination. We screened polymers from a library of beta-amino esters for their ability to augment transgene expression as measured by beta-galactosidase activity and cellular immune responses. Among the candidates identified in this screen, poly[(1,6-di(acryloxyethoxy)hexane)-co-(4-aminobutanol)] enhanced plasmid DNA transgene expression by sevenfold (P=0.0001) and its immunogenicity by 70% (P=0.03). We found that polymers with moderately hydrophobic backbones and terminal alcohol groups facilitated transfection most effectively in vivo. We also observed a log-linear correlation (R2=0.93) between peak cellular immune responses and transgene activity in all evaluated polymer-plasmid DNA formulations, clarifying the relationship between immunogenicity and the quantity of expressed antigen.     

Analysis of Gag-specific Cytotoxic T Lymphocytes in Simian Immunodeficiency Virus–infected Rhesus Monkeys by Cell Staining with a Tetrameric Major Histocompatibility Complex Class I–Peptide Complex

A tetrameric recombinant major histocompatibility complex (MHC) class I–peptide complex was used as a staining reagent in flow cytometric analyses to quantitate and define the phenotype of Gag-specific cytotoxic T lymphocytes (CTLs) in the peripheral blood of simian immunodeficiency virus macaque (SIVmac)-infected rhesus monkeys. The heavy chain of the rhesus monkey MHC class I molecule Mamu-A*01 and β₂-microglobulin were refolded in the presence of an SIVmac Gag synthetic peptide (p11C, C–M) representing the optimal nine–amino acid peptide of Mamu-A*01–restricted predominant CTL epitope to create a tetrameric Mamu-A*01/p11C, C–M complex. Tetrameric Mamu-A*01/p11C, C–M complex bound to T cells of SIVmac-infected, Mamu-A*01⁺, but not uninfected, Mamu-A*01⁺, or infected, Mamu-A*01⁻ rhesus monkeys. Specific staining of peripheral blood mononuclear cells (PBMC) from SIVmac-infected, Mamu-A*01⁺ rhesus monkeys was only found in the cluster of differentiation (CD)8α/β⁺ T lymphocyte subset and the percentage of CD8α/β⁺ T cells in the peripheral blood of four SIVmac-infected, Mamu-A*01⁺ rhesus monkeys staining with this complex ranged from 0.7 to 10.3%. Importantly, functional SIVmac Gag p11C-specific CTL activity was seen in sorted and expanded tetrameric Mamu-A*01/p11C, C–M complex–binding, but not nonbinding, CD8α/β⁺ T cells. Furthermore, the percentage of CD8α/β⁺ T cells binding this tetrameric Mamu-A*01/p11C, C–M complex correlated well with p11C-specific cytotoxic activity as measured in both bulk and limiting dilution effector frequency assays. Finally, phenotypic characterization of the cells binding this tetrameric complex indicated that this lymphocyte population is heterogeneous. These studies indicate the power of this approach for examining virus-specific CTLs in in vivo settings.Cytotoxic T lymphocytes (CTLs) play an important role in containing virus spread in many viral infections. However, the activity of this cell population in vivo has proven difficult to study because its evaluation has relied on cumbersome, functional assays that require extensive cell manipulation and lengthy in vitro periods of cell cultivation. Altman et al. have recently reported that fluorescence dye-coupled tetrameric MHC class I–peptide complexes can specifically bind to subpopulations of epitope-specific cluster of differentiation (CD)¹8⁺ T cells, raising the possibility that CTLs might be studied using flow cytometric technology (1).There is accumulating evidence for the importance of CTLs in controlling HIV-1 and simian immunodeficiency virus replication in both primary and chronic infections (2– 6). We have been studying the role of this cellular immune response in AIDS immunopathogenesis in the simian immunodeficiency virus (SIV)/macaque model of AIDS. Much of this work has focused on the evaluation of SIVmac Gag recognition by CTL in rhesus monkeys expressing the HLA-A homologue molecule Mamu-A*01. In fact, we have shown that CTL recognition of Gag in SIVmac-infected or vaccinated Mamu-A*01⁺ rhesus monkeys is restricted to a single epitope, 12–amino acid fragment of SIVmac 251 Gag (amino acid 179–190) (p11C), bound to Mamu-A*01 (7). Through studying the monkeys'' response to this dominant CTL epitope, we have been able to evaluate efficiently a variety of novel vaccine strategies for eliciting SIVmac-specific CTL responses and assess the role of CTLs in containing the replication of SIVmac during primary and chronic infections (8–11).In these studies, we have generated tetrameric Mamu-A*01/p11C, C–M complex using the optimal nine–amino acid fragment of SIVmac (amino acids 181–189) p11C, C-M (12) and evaluated its binding specificity in PBMCs of SIVmac-infected, Mamu-A*01⁺ rhesus monkeys. We demonstrate that the enumeration of CD8⁺ T cells that bind this complex in flow cytometric analyses correlates quantitatively with functional CTL activity and that this cell population is phenotypically heterogeneous.