Recombinant HIV-1 vaccine candidates based on replication-defective flavivirus vector.

Multiple approaches utilizing viral and DNA vectors have shown promise in the development of an effective vaccine against HIV. In this study, an alternative replication-defective flavivirus vector, RepliVax (RV), was evaluated for the delivery of HIV-1 immunogens. Recombinant RV-HIV viruses were engineered to stably express clade C virus Gag and Env (gp120TM) proteins and propagated in Vero helper cells. RV-based vectors enabled efficient expression and correct maturation of Gag and gp120TM proteins, were apathogenic in a sensitive suckling mouse neurovirulence test, and were similar in immunogenicity to recombinant poxvirus NYVAC-HIV vectors in homologous or heterologous prime-boost combinations in mice. In a pilot NHP study, immunogenicity of RV-HIV viruses used as a prime or boost for DNA or NYVAC candidates was compared to a DNA prime/NYVAC boost benchmark scheme when administered together with adjuvanted gp120 protein. Similar neutralizing antibody titers, binding IgG titers measured against a broad panel of Env and Gag antigens, and ADCC responses were observed in the groups throughout the course of the study, and T cell responses were elicited. The entire data demonstrate that RV vectors have the potential as novel HIV-1 vaccine components for use in combination with other promising candidates to develop new effective vaccination strategies.

The impact of HLA class I and EBV latency-II antigen-specific CD8(+) T cells on the pathogenesis of EBV(+) Hodgkin lymphoma.

In 40% of cases of classical Hodgkin lymphoma (cHL), Epstein-Barr virus (EBV) latency-II antigens [EBV nuclear antigen 1 (EBNA1)/latent membrane protein (LMP)1/LMP2A] are present (EBV(+) cHL) in the malignant cells and antigen presentation is intact. Previous studies have shown consistently that HLA-A*02 is protective in EBV(+) cHL, yet its role in disease pathogenesis is unknown. To explore the basis for this observation, gene expression was assessed in 33 cHL nodes. Interestingly, CD8 and LMP2A expression were correlated strongly and, for a given LMP2A level, CD8 was elevated markedly in HLA-A*02(-) versus HLA-A*02(+) EBV(+) cHL patients, suggesting that LMP2A-specific CD8(+) T cell anti-tumoral immunity may be relatively ineffective in HLA-A*02(-) EBV(+) cHL. To ascertain the impact of HLA class I on EBV latency antigen-specific immunodominance, we used a stepwise functional T cell approach. In newly diagnosed EBV(+) cHL, the magnitude of ex-vivo LMP1/2A-specific CD8(+) T cell responses was elevated in HLA-A*02(+) patients. Furthermore, in a controlled in-vitro assay, LMP2A-specific CD8(+) T cells from healthy HLA-A*02 heterozygotes expanded to a greater extent with HLA-A*02-restricted compared to non-HLA-A*02-restricted cell lines. In an extensive analysis of HLA class I-restricted immunity, immunodominant EBNA3A/3B/3C-specific CD8(+) T cell responses were stimulated by numerous HLA class I molecules, whereas the subdominant LMP1/2A-specific responses were confined largely to HLA-A*02. Our results demonstrate that HLA-A*02 mediates a modest, but none the less stronger, EBV-specific CD8(+) T cell response than non-HLA-A*02 alleles, an effect confined to EBV latency-II antigens. Thus, the protective effect of HLA-A*02 against EBV(+) cHL is not a surrogate association, but reflects the impact of HLA class I on EBV latency-II antigen-specific CD8(+) T cell hierarchies.

A mine is a terrible thing to waste: high content, single cell technologies for comprehensive immune analysis.

In recent years, an incredible variety of single cell technologies have become available to analyze immune responses. These technologies include polychromatic flow cytometry, mass cytometry, highly multiplexed single cell qPCR, RNA sequencing, microtools, and high-resolution imaging. In this article, we review these platforms, describing their power and limitations for comprehensive analysis of the immune system. We relate the properties of these technologies to the various cellular states relevant to an immune response, in order to address which technologies are most appropriate for which settings.

Comparison of systemic and mucosal vaccination: impact on intravenous and rectal SIV challenge.

Mucosal tissues are the primary route of transmission for most respiratory and sexually transmitted diseases, including human immunodeficiency virus. We aimed to generate strong mucosal immune responses to simian immunodeficiency virus (SIV) in rhesus macaques by targeting recombinant adenovirus serotype 5 (rAd5) to the lung. The immunogenicity and efficacy of aerosol (AE) vaccination was compared with intramuscular (IM) delivery in either an intravenous (IV) or intrarectal (IR) SIV(mac251) challenge model. Aerosolized rAd5 induced strong cellular responses in the lung and systemic humoral responses equivalent to IM. Strikingly, all immunization groups controlled acute viremia in the IV challenge model by 1-2 logs. By contrast, after IR challenge, only peak viremia was reduced by immunization, with no significant effect on SIV infection acquisition rate or mucosal CD4(+) T-cell preservation. Improved disease outcome was associated with pre-challenge cellular and humoral responses, while post-challenge T-cell responses were highly correlated with viremia control. The similar outcomes achieved by systemic and airway mucosal immunization support AE delivery as a safe, effective, and less invasive alternative to parenteral vaccination.

A replication defective recombinant Ad5 vaccine expressing Ebola virus GP is safe and immunogenic in healthy adults.

Ebola virus causes irregular outbreaks of severe hemorrhagic fever in equatorial Africa. Case mortality remains high; there is no effective treatment and outbreaks are sporadic and unpredictable. Studies of Ebola virus vaccine platforms in non-human primates have established that the induction of protective immunity is possible and safety and human immunogenicity has been demonstrated in a previous Phase I clinical trial of a 1st generation Ebola DNA vaccine. We now report the safety and immunogenicity of a recombinant adenovirus serotype 5 (rAd5) vaccine encoding the envelope glycoprotein (GP) from the Zaire and Sudan Ebola virus species, in a randomized, placebo-controlled, double-blinded, dose escalation, Phase I human study. Thirty-one healthy adults received vaccine at 2×10(9) (n=12), or 2×10(10) (n=11) viral particles or placebo (n=8) as an intramuscular injection. Antibody responses were assessed by ELISA and neutralizing assays; and T cell responses were assessed by ELISpot and intracellular cytokine staining assays. This recombinant Ebola virus vaccine was safe and subjects developed antigen specific humoral and cellular immune responses.

Publishing flow cytometry data.

Cellular measurements by flow cytometric analysis constitute an important step toward understanding individual attributes within a population of cells. Assessing individual cells within a population by protein expression using fluorescently labeled antibodies and other fluorescent probes can identify cellular patterns. The technology for accurately identifying subtle changes in protein expression within a population of cells using a vast array of technology has resulted in controversy and questions regarding reproducibility, which can be explained at least in part by the absence of standard methods to facilitate comparison of flow cytometric data. The complexity of technological advancements and the need for improvements in biological resolution results in the generation of complex data that demands the use of minimum standards for their publication. Herein we present a summarized view for the inclusion of consistent flow cytometric experimental information as supplemental data. Four major points, experimental and sample information, data acquisition, analysis, and presentation are emphasized. Together, these guidelines will facilitate the review and publication of flow cytometry data that provide an accurate foundation for ongoing studies with this evolving technology.

CD4 T cell subsets in the mucosa are CD28+Ki-67-HLA-DR-CD69+ but show differential infection based on alpha4beta7 receptor expression during acute SIV infection.

BACKGROUND: CD4 T cell depletion in the mucosa has been well documented during acute HIV and SIV infections. The demonstration the HIV/SIVcan use the alpha4beta7 receptor for viral entry suggests that these viruses selectively target CD4 T cells in the mucosa that express high levels of alpha4beta7 receptor. METHODS: Mucosal samples obtained from SIV infected rhesus macaques during the early phase of infection were used for immunophenotypic analysis. CD4 T cell subsets were sorted based on the expression of beta7 and CD95 to quantify the level of SIV infection in different subsets of CD4 T cells. Changes in IL-17, IL-21, IL-23 and TGFbeta mRNA expression was determined using Taqman PCR. RESULTS: CD4 T cells in the mucosa were found to harbor two major population of cells; -25% of CD4 T cells expressed the alpha4(+)beta7(hi) phenotype, whereas the rest of the 75% expressed an alpha4(+)beta7(int) phenotype. Both the subsets were predominantly CD28(+)Ki-67(-)HLA-DR(-) but CD69(+), and expressed detectable levels of CCR5 on their surface. Interestingly, however, alpha4(+)beta7(hi)CD4 T cells were found to harbor more SIV than the alpha4(+)beta7(int) subsets at day 10 pi. Early infection was associated with a dramatic increase in the expression of IL-17, and IL-17 promoting cytokines IL-21, IL-23, and TGFbeta that stayed high even after the loss of mucosal CD4 T cells. CONCLUSIONS: Our results suggest that the differential expression of the alpha4beta7 receptor contributes to the differences in the extent of infection in CD4 T cell subsets in the mucosa. Early infection is associated dysregulation of the IL-17 network in mucosal tissues involves other non-Th-17 cells that likely contributes to the pro-inflammatory environment in the mucosa during acute stages of SIV infection.

Alpha4(+)beta7(hi)CD4(+) memory T cells harbor most Th-17 cells and are preferentially infected during acute SIV infection.

Human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) infections are believed to infect minimally activated CD4(+) T cells after viral entry. Not much is known about why SIV selectively targets these cells. Here we show that CD4(+) T cells that express high levels of the alpha4beta7 heterodimer are preferentially infected very early during the course of SIV infection. At days 2-4 post infection, alpha4(+)beta7(hi)CD4(+) T cells had approximately 5x more SIV-gag DNA than beta7(-)CD4(+) T cells. alpha4(+)beta7(hi)CD4(+) T cells displayed a predominantly central memory (CD45RA(-)CD28(+)CCR7(+)) and a resting (CD25(-)CD69(-)HLA-DR(-)Ki-67(-)) phenotype. Although the expression of detectable CCR5 was variable on alpha4(+)beta7(hi) and beta7(-)CD4(+) T cells, both CCR5(+) and CCR5(-) subsets of alpha4(+)beta7(hi) and beta7(-)CD4(+) T cells were found to express sufficient levels of CCR5 mRNA, suggesting that both these subsets could be efficiently infected by SIV. In line with this, we found similar levels of SIV infection in beta7(-)CD4(+)CCR5(+) and beta7(-)CD4(+)CCR5(-) T cells. alpha4beta7(hi)CD4(+) T cells were found to harbor most T helper (Th)-17 cells that were significantly depleted during acute SIV infection. Taken together, our results show that resting memory alpha4(+)beta7(hi)CD4(+) T cells in the blood are preferentially infected and depleted during acute SIV infection, and the loss of these cells alters the balance between Th-17 and Th-1 responses, thereby contributing to disease pathogenesis.

Spectral compensation for flow cytometry: visualization artifacts, limitations, and caveats.

BACKGROUND: In multicolor flow cytometric analysis, compensation for spectral overlap is nearly always necessary. For the most part, such compensation has been relatively simple, producing the desired rectilinear distributions. However, in the realm of multicolor analysis, visualization of compensated often results in unexpected distributions, principally the appearance of a large number of events on the axis, and even more disconcerting, an inability to bring the extent of compensated data down to "autofluorescence" levels. MATERIALS AND METHODS: A mathematical model of detector measurements with variable photon intensities, spillover parameters, measurement errors, and data storage characteristics was used to illustrate sources of apparent error in compensated data. Immunofluorescently stained cells were collected under conditions of limiting light collection and high spillover between detectors to confirm aspects of the model. RESULTS: Photon-counting statistics contribute a nonlinear error to compensated parameters. Measurement errors and log-scale binning error contribute linear errors to compensated parameters. These errors are most apparent with the use of red or far-red fluorochromes (where the emitted light is at low intensity) and with large spillover between detectors. Such errors can lead to data visualization artifacts that can easily lead to incorrect conclusions about data, and account for the apparent "undercompensation" previously described for multicolor staining. CONCLUSIONS: There are inescapable errors arising from imperfect measurements, photon-counting statistics, and even data storage methods that contribute both linearly and nonlinearly to a "spreading" of a properly compensated autofluorescence distribution. This phenomenon precludes the use of "quadrant" statistics or gates to analyze affected data; it also precludes visual adjustment of compensation. Most importantly, it is impossible to properly compensate data using standard visual graphical interfaces (histograms or dot plots). Computer-assisted compensation is required, as well as careful gating and experimental design to determine the distinction between positive and negative events. Finally, the use of special staining controls that employ all reagents except for the one of interest (termed fluorescence minus one, or "FMO" controls) becomes necessary to accurately identify expressing cells in the fully stained sample.

Probability binning comparison: a metric for quantitating univariate distribution differences.

BACKGROUND: Comparing distributions of data is an important goal in many applications. For example, determining whether two samples (e.g., a control and test sample) are statistically significantly different is useful to detect a response, or to provide feedback regarding instrument stability by detecting when collected data varies significantly over time. METHODS: We apply a variant of the chi-squared statistic to comparing univariate distributions. In this variant, a control distribution is divided such that an equal number of events fall into each of the divisions, or bins. This approach is thereby a mini-max algorithm, in that it minimizes the maximum expected variance for the control distribution. The control-derived bins are then applied to test sample distributions, and a normalized chi-squared value is computed. We term this algorithm Probability Binning. RESULTS: Using a Monte-Carlo simulation, we determined the distribution of chi-squared values obtained by comparing sets of events derived from the same distribution. Based on this distribution, we derive a conversion of any given chi-squared value into a metric that is analogous to a t-score, i.e., it can be used to estimate the probability that a test distribution is different from a control distribution. We demonstrate that this metric scales with the difference between two distributions, and can be used to rank samples according to similarity to a control. Finally, we demonstrate the applicability of this metric to ranking immunophenotyping distributions to suggest that it indeed can be used to objectively determine the relative distance of distributions compared to a single control. CONCLUSION: Probability Binning, as shown here, provides a useful metric for determining the probability that two or more flow cytometric data distributions are different. This metric can also be used to rank distributions to identify which are most similar or dissimilar. In addition, the algorithm can be used to quantitate contamination of even highly-overlapping populations. Finally, as demonstrated in an accompanying paper, Probability Binning can be used to gate on events that represent significantly different subsets from a control sample. Published 2001 Wiley-Liss, Inc.

Probability binning comparison: a metric for quantitating multivariate distribution differences.

BACKGROUND: While several algorithms for the comparison of univariate distributions arising from flow cytometric analyses have been developed and studied for many years, algorithms for comparing multivariate distributions remain elusive. Such algorithms could be useful for comparing differences between samples based on several independent measurements, rather than differences based on any single measurement. It is conceivable that distributions could be completely distinct in multivariate space, but unresolvable in any combination of univariate histograms. Multivariate comparisons could also be useful for providing feedback about instrument stability, when only subtle changes in measurements are occurring. METHODS: We apply a variant of Probability Binning, described in the accompanying article, to multidimensional data. In this approach, hyper-rectangles of n dimensions (where n is the number of measurements being compared) comprise the bins used for the chi-squared statistic. These hyper-dimensional bins are constructed such that the control sample has the same number of events in each bin; the bins are then applied to the test samples for chi-squared calculations. RESULTS: Using a Monte-Carlo simulation, we determined the distribution of chi-squared values obtained by comparing sets of events from the same distribution; this distribution of chi-squared values was identical as for the univariate algorithm. Hence, the same formulae can be used to construct a metric, analogous to a t-score, that estimates the probability with which distributions are distinct. As for univariate comparisons, this metric scales with the difference between two distributions, and can be used to rank samples according to similarity to a control. We apply the algorithm to multivariate immunophenotyping data, and demonstrate that it can be used to discriminate distinct samples and to rank samples according to a biologically-meaningful difference. CONCLUSION: Probability binning, as shown here, provides a useful metric for determining the probability with which two or more multivariate distributions represent distinct sets of data. The metric can be used to identify the similarity or dissimilarity of samples. Finally, as demonstrated in the accompanying paper, the algorithm can be used to gate on events in one sample that are different from a control sample, even if those events cannot be distinguished on the basis of any combination of univariate or bivariate displays. Published 2001 Wiley-Liss, Inc.

Frequency difference gating: a multivariate method for identifying subsets that differ between samples.

BACKGROUND: In multivariate distributions (for example, in 3- or more color flow cytometric datasets), it can become difficult or impossible to identify populations that differ between samples based only on a combination of univariate or bivariate displays. Indeed, it is possible that such differences can only be identified in "n"-dimensional space, where "n" is the number of parameters measured. Therefore, computer assisted identification of such differences is necessary. Such a method could be used to identify responses (i.e., by comparing cell samples before and after stimulation) in exquisite detail by allowing complete analysis of the collected data on only those events which have responded. METHODS: Multivariate Probability Binning can be used to compare different datasets to identify the distance and statistical significance of a difference between the distributions. An intermediate step in the algorithm provides access to the actual locations within the n-dimensional comparison which are most different between the distributions. Gates based on collections of hyper-rectangular bins can then be applied to datasets, thereby selecting those events (or clusters of events) that are different between samples. We term this process Frequency Difference Gating. RESULTS: Frequency Difference Gating was used in several test scenarios to evaluate its utility. First, we compared PBMC subsets identified by solely by immunofluorescence staining: based on this training data set, the algorithm automatically generated an accurate forward and side-scatter gate to identify lymphocytes. Second, we applied the algorithm to identify subtle differences between CD4 memory subsets based on 8-color immunophenotyping data. The resulting 3-dimensional gate could resolve cells subsets much more frequent in one subset compared to the other; no combination of two-dimensional gates could accomplish this resolution. Finally, we used the algorithm to compare B cell populations derived from mice of different ages or strains, and found that the algorithm could find very subtle differences between the populations. CONCLUSION: Frequency Difference Gating is a powerful tool that automates the process of identifying events comprising underlying differences between samples. It is not a clustering tool; it is not meant to identify subsets in multidimensional space. Importantly, this method may reveal subtle changes in small populations of cells, changes that only occur simultaneously in multiple dimensions in such a way that identification by univariate or bivariate analyses is impossible. Finally, the method may significantly aid in the analysis of high-order multivariate data (i.e., 6-12 color flow cytometric analyses), where identification of differences between datasets becomes so time-consuming as to be impractical. Published 2001 Wiley-Liss, Inc.

Vdelta1 and Vdelta2 gammadelta T cells express distinct surface markers and might be developmentally distinct lineages.

We report here that the two major types of gammadelta T cells found in human blood, Vdelta1 and Vdelta2, were found to have markedly different phenotypes. Vdelta2 cells had a phenotype typical of most alphabeta T cells in blood; i.e., they were CD5(+), CD28(+), and CD57(-). In contrast, Vdelta1 cells tended to be CD5(-/dull), CD28(-), and CD57(+). Furthermore, although Vdelta1 T cells appeared to be "naive" in that they were CD45RA(+), they were CD62L(-) and on stimulation uniformly produced interferon-gamma, indicating that they are in fact memory/effector cells. This phenotype for Vdelta1 cells was similar to that of intestinal intraepithelial lymphocytes, a subset that can develop in the absence of the thymus. We suggest that the Vdelta1 and Vdelta2 T cell subsets represent distinct lineages with different developmental pathways. The disruption of the supply of normal, thymus-derived T cells in HIV-infected individuals might be responsible for the shift in the Vdelta2/Vdelta1 ratio that occurs in the blood of individuals with HIV disease.

HIV-1 actively replicates in naive CD4(+) T cells residing within human lymphoid tissues.

Although HIV-1 gene expression is detected in naive, resting T cells in vivo, such cells are resistant to productive infection in vitro. However, we found that the endogenous microenvironment of human lymphoid tissues supports de novo infection and depletion of this population. Cell cycle analysis and DNA labeling experiments established that these cells were definitively quiescent and thus infected de novo. Quantitation of the "burst size" within naive cells further demonstrated that these cells were productively infected and contributed to the local viral burden. These findings demonstrate that lymphoid tissues support active HIV-1 replication in resting, naive T cells. Moreover, these cells are not solely reservoirs of latent virus but are permissive hosts for viral replication that likely targets them for elimination.

Naive CD4 T cells inhibit CD28-costimulated R5 HIV replication in memory CD4 T cells.

Stimulation with antibodies to CD3 and CD28 coimmobilized on beads can be used to significantly expand T cells ex vivo. With CD4 T cells from HIV-infected patients, this expansion usually is accompanied by complete suppression of viral replication, presumed to be caused by down-regulation of the viral coreceptor CCR5 and up-regulation of CCR5 ligands. Here we show that this suppression occurs in total CD4 T cells acutely infected with R5 HIV, but not in purified CD62L(-) memory CD4 T cells. The lack of complete suppression in these memory cells, typically comprising 10-40% of total CD4 T cells, occurs despite high levels of CCR5 ligand secretion and down-regulation of CCR5. Significantly, adding back naive or CD62L(+) memory CD4 T cells inhibits the viral replication in the CD62L(-) cells, with the naive cells capable of completely repressing the virus. Although this inhibition was previously thought to be specific to bead-bound anti-CD3/CD28 stimulation, we show that the same suppression is obtained with sufficiently strong anti-CD3/B7.1 stimulation. Our results show that inhibitory mechanisms, expressed predominantly by strongly stimulated naive CD4 T cells and mediated independently of CCR5-binding chemokines, play a role in the inhibition of R5 HIV replication in CD4 T cells upon CD28 costimulation.

Phycoerythrin-allophycocyanin: a resonance energy transfer fluorochrome for immunofluorescence.

BACKGROUND: As immunofluorescence experiments become more complex, the demand for new dyes with different properties increases. Fluorescent dyes with large Stoke's shifts that are very bright and have low background binding to cells are especially desirable. We report on the properties of the resonance energy tandems of phycoerythrin and allophycocyanin (PE-APC). PE-APC is the original fluorescence resonance energy tandem dye described in the literature, but it has not been utilized because of the difficulty of synthesizing and preparing a consistent product. METHODS: PE-APC complexes comprising different ratios of the two phycobiliproteins conjugated to streptavidin were synthesized using standard protein-protein conjugation chemistry. The PE-APC streptavidins were evaluated for flow cytometric analysis. They were compared directly to Cy5PE conjugates because Cy5PE is the fluorophore that is spectrally most like the PE-APC. RESULTS: PE-APC complexes showed the expected fluorescence spectral properties of a tandem: excitation was excellent at 488 nm (and best at the PE excitation maximum) and emission was greatest at the APC emission maximum at about 660 nm. The efficiency of transfer of energy from PE to APC was about 90%. CONCLUSION: PE-APC can be considered an excellent substitute for Cy5PE. Compared with Cy5PE, PE-APC has similar brightness (in staining experiments), slightly greater compensation requirements with PE but much lower compensation with Cy5.5PE or Cy5.5PerCP, and lower nonspecific background binding. PE-APC is a useful alternative to Cy5PE, especially in applications in which the use of Cy5 is impractical. Cytometry 44:24-29, 2001. Published 2001 Wiley-Liss, Inc.

Direct functional analysis of epitope-specific CD8+ T cells in peripheral blood.

The functional status of virus-specific CD8+ T cells is important for the outcome and the immunopathogenesis of viral infections. We have developed an assay for the direct functional analysis of antigen-specific CD8+ T cells, which does not require prolonged in vitro cultivation and amplification of T cells. Whole blood samples were incubated with peptide antigens for <5 h, followed by staining with peptide-MHC tetramers to identify epitope-specific T cells. The cells were also stained for the activation marker CD69 or for the production of cytokines such as interferon-gamma (IFNgamma) or tumor necrosis factor-alpha (TNFalpha). With the combined staining with tetramer and antibodies to CD69 or cytokines the number of antigen-specific CD8+ T cells as well as the functional response of each individual cell to the cognate antigen can be determined in a single experiment. Virus-specific CD8+ T cells that are nonfunctional, as well as those that are functional under the same stimulating conditions can be simultaneously detected with this assay, which is not possible by using other T-cell functional assays including cytotoxicity assay, intracellular cytokine staining, and enzyme-linked immunospot (ELISPOT) assay.