Joseph Casazza - Academia.edu (original) (raw)

Papers by Joseph Casazza

Research paper thumbnail of Purification, characterization and initial rate kinetics of acyl-phosphate-hexose phosphotransferase from Aerobacter aerogenes

Research paper thumbnail of Hydroxylation of acetone by ethanol-inducible P-450 isozyme 3a

Federation Proceedings, 1985

Research paper thumbnail of Early Depletion of<i>Mycobacterium tuberculosis</i>–Specific T Helper 1 Cell Responses after HIV‐1 Infection

The Journal of Infectious Diseases, Dec 1, 2008

Background. The acid-fast bacillus Mycobacterium tuberculosis is often the first manifestation of... more Background. The acid-fast bacillus Mycobacterium tuberculosis is often the first manifestation of acquired immunodeficiency syndrome in patients infected with human immunodeficiency virus (HIV). This study was conducted to better understand the mechanism underlying M. tuberculosis-specific pathogenicity early after onset of HIV infection. Methods. M. tuberculosis-specific T helper 1 (Th1) cells were studied in HIV negative (n ϭ 114) and chronically HIV infected (n ϭ 68) Tanzanian subjects by using early secreted antigenic target 6 (ESAT6) protein or tuberculin (purified protein derivative) with interferon-␥ ELISPOT and intracellular cytokine staining. In a longitudinal study, the effect of acute HIV infection on M. tuberculosis-specific Th1 cells was determined by polychromatic flow cytometric analysis in 5 subjects with latent M. tuberculosis infection who became infected with HIV. Results. In tuberculosis (TB)-asymptomatic subjects (i.e., subjects with unknown TB status who did not show clinical signs suggestive of TB), chronic HIV infection was associated with a decreased percentage of subjects with detectable M. tuberculosis-specific Th1 cells (P Ͻ .001), a decrease which was not observed among subjects with active TB. Acute HIV infection induced a rapid depletion of M. tuberculosis-specific Th1 cells in 4 subjects remained TB asymptomatic, whereas the population of these cells remained stable in subjects who remained HIV negative (P Ͻ .01). Conclusions. Taken together, these data suggest a mechanism of rapid M. tuberculosis-specific Th1 cell depletion that may contribute to the early onset of TB in individuals with latent M. tuberculosis infection who become HIV infected.

Research paper thumbnail of TCR recognition is a mediator in the control of HIV infection by CD8 T cells

Journal of Immunology, May 1, 2017

Research paper thumbnail of Trispecific antibody targeting HIV-1 and T cells activates and eliminates latently-infected cells in HIV/SHIV infections

Nature Communications

Agents that can simultaneously activate latent HIV, increase immune activation and enhance the ki... more Agents that can simultaneously activate latent HIV, increase immune activation and enhance the killing of latently-infected cells represent promising approaches for HIV cure. Here, we develop and evaluate a trispecific antibody (Ab), N6/αCD3-αCD28, that targets three independent proteins: (1) the HIV envelope via the broadly reactive CD4-binding site Ab, N6; (2) the T cell antigen CD3; and (3) the co-stimulatory molecule CD28. We find that the trispecific significantly increases antigen-specific T-cell activation and cytokine release in both CD4+ and CD8+ T cells. Co-culturing CD4+ with autologous CD8+ T cells from ART-suppressed HIV+ donors with N6/αCD3-αCD28, results in activation of latently-infected cells and their elimination by activated CD8+ T cells. This trispecific antibody mediates CD4+ and CD8+ T-cell activation in non-human primates and is well tolerated in vivo. This HIV-directed antibody therefore merits further development as a potential intervention for the eradicati...

Research paper thumbnail of An influenza hemagglutinin stem nanoparticle vaccine induces cross-group 1 neutralizing antibodies in healthy adults

Science Translational Medicine

Influenza vaccines could be improved by platforms inducing cross-reactive immunity. Immunodominan... more Influenza vaccines could be improved by platforms inducing cross-reactive immunity. Immunodominance of the influenza hemagglutinin (HA) head in currently licensed vaccines impedes induction of cross-reactive neutralizing stem-directed antibodies. A vaccine without the variable HA head domain has the potential to focus the immune response on the conserved HA stem. This first-in-human dose-escalation open-label phase 1 clinical trial (NCT03814720) tested an HA stabilized stem ferritin nanoparticle vaccine (H1ssF) based on the H1 HA stem of A/New Caledonia/20/1999. Fifty-two healthy adults aged 18 to 70 years old enrolled to receive either 20 μg of H1ssF once ( n = 5) or 60 μg of H1ssF twice ( n = 47) with a prime-boost interval of 16 weeks. Thirty-five (74%) 60-μg dose participants received the boost, whereas 11 (23%) boost vaccinations were missed because of public health restrictions in the early stages of the COVID-19 pandemic. The primary objective of this trial was to evaluate ...

Research paper thumbnail of Potent anti-viral activity of a trispecific HIV neutralizing antibody in SHIV-infected monkeys

Cell Reports

Broadly neutralizing antibodies (bNAbs) represent an alternative to drug therapy for the treatmen... more Broadly neutralizing antibodies (bNAbs) represent an alternative to drug therapy for the treatment of HIV-1 infection. Immunotherapy with single bNAbs often leads to emergence of escape variants, suggesting a potential benefit of combination bNAb therapy. Here, a trispecific bNAb reduces viremia 100- to 1000-fold in viremic SHIV-infected macaques. After treatment discontinuation, viremia rebounds transiently and returns to low levels, through CD8-mediated immune control. These viruses remain sensitive to the trispecific antibody, despite loss of sensitivity to one of the parental bNAbs. Similarly, the trispecific bNAb suppresses the emergence of resistance in viruses derived from HIV-1-infected subjects, in contrast to parental bNAbs. Trispecific HIV-1 neutralizing antibodies, therefore, mediate potent antiviral activity in vivo and may minimize the potential for immune escape.

Research paper thumbnail of Application of B cell immortalization for the isolation of antibodies and B cell clones from vaccine and infection settings

The isolation and characterization of neutralizing antibodies from infection and vaccine settings... more The isolation and characterization of neutralizing antibodies from infection and vaccine settings will inform future vaccine design, and methodologies that streamline the isolation of antibodies and the generation of B cell clones are of great interest. Retroviral transduction to express Bcl-6 and Bcl-xL in primary B cells has been shown to promote long-term B cell survival and antibody secretion in vitro, and can be used to isolate antibodies from memory B cells. The application of this methodology to B cell subsets from tissues and to B cells from individuals with chronic infection has not been extensively characterized. Here, we characterize Bcl-6/Bcl-xL B cell immortalization across multiple tissue types and B cell subsets in healthy and HIV-1 infected individuals, as well as individuals recovering from malaria. In HIV-1- and malaria-uninfected donors, naïve and memory B cell subsets from PBMC and tonsil tissue transformed with similar efficiencies, and displayed similar charact...

Research paper thumbnail of Th1/Th17 cell induction following vaccination with the novel tuberculosis vaccine MVA85A and their role in Mycobacterium tuberculosis challengeCD4 T cell response to MVA85A vaccination against Mycobacterium tuberculosis in healthy HIV-infected adults SO IMMUNOLOGY CT Annual Congress of the Britis...

Research paper thumbnail of CD4 T cell response to MVA85A vaccination against Mycobacterium tuberculosis in healthy HIV-infected adults

Research paper thumbnail of Sustained HIV release by single persisting CD4 + T cells during latency disruption

Journal of Virus Eradication, 2015

cells had returned to resting. Second, cell proliferation of acutely infected cultures was tracke... more cells had returned to resting. Second, cell proliferation of acutely infected cultures was tracked using CFSE dye and cell subsets that had undergone varying degrees of proliferation were isolated by FACS, at the end of culture. Each recovered cell subset was analyzed for the quantity of integrated HIV DNA and replication-competent virus. Results: Cells exposed to HIV, prior to stimulation, contained the highest levels of integrated provirus and replication competent virus after returning to quiescence. Cells infected, during the height of cell proliferation, retained the least HIV. Cells that did not divide or divided only a few times contained higher levels of integrated and replicationcompetent HIV than did cells, which had divided many times. Conclusions: HIV latency is established very early within a heterogeneous population of CD4 T cells, which are undergoing varying degrees of cell activation in the presence of infectious virus. Minimally activated cells within this milieu are the most likely to develop persistent latent infection. These observations have important implications for the study of latency, and may aid in the design of strategies to purge this reservoir.

Research paper thumbnail of Uncovering mechanisms of HIV persistence in HIV controllers by HIV sequence analysis in CD4 T cell subsets

Journal of Virus Eradication, 2015

or more of these latency-reversing agents (LRAs) will be combined with a vaccine capable of elici... more or more of these latency-reversing agents (LRAs) will be combined with a vaccine capable of eliciting cytotoxic T cells to clear reactivated cells. Therefore, we studied the effects of these drugs on T cells. Methods: We examined the in vitro impact of clinically-relevant exposures to putative LRAs on T cell activation and function. Histone deacetylase inhibitors (HDACi) vorinostat (VOR), panobinostat (PAN) and romidepsin (ROMI) were compared with protein kinase C modulators ingenol 3,20 dibenzoate (ING), prostratin (PRO) and bryostatin-1 (BRYO). Results: No effect of VOR was observed on T cell activation, whereas PAN and ROMI consistently increased expression of CD69 on CD4 and CD8 T cells. PKC modulators all induced increased expression of CD69 and MHC I on CD4 and CD8 T cells, which for CD69 was sustained over 72 hours. VOR and ROMI did not impact ex vivo antigen-specific CD8 T cell responses as measured by degranulation and cytokine production six hours after stimulation. PAN, however, produced a small but significant decrease in the frequency of antigen-specific CD8 T cell responses. PKC modulators all induced non-specific cytokine production, an effect that was more marked in HIV seropositive than in seronegative individuals, but did not significantly affect antigen-specific responses. Interestingly, ROM, PRO and BRYO inhibited antigen-dependent proliferation over a 5 day in vitro culture in both seropositive and seronegative participants. PRO and BRYO were toxic in these short term culture assays. Conclusions: Even within the same class, LRAs vary in their effect on the phenotypic profile and functional capacity of T cells. Moreover, effects on T cell function may not be detectable until several days after dosing. These differences should be considered for dosing, designing and testing of HIV latency clearance strategies.

Research paper thumbnail of Pretranslational activation of cytochrome P450IIE during ketosis induced by a high fat diet

PubMed, Mar 1, 1992

Ethanol-inducible cytochrome P450 (P450IIE) is reported to be induced by ketosis. In the present ... more Ethanol-inducible cytochrome P450 (P450IIE) is reported to be induced by ketosis. In the present study, the effects of a high fat diet on P450IIE induction and the relationship between ketone body concentration and P450IIE induction were studied by the following: 1) measurement of the activity of aniline hydroxylase, 2) immunoblot analysis for P450IIE protein, and 3) Northern blot analysis for P450IIE mRNA. The enzyme activities (aniline hydroxylase) in hepatic and renal microsomes were elevated about 2-3-fold by feeding with a high fat diet for 3 days. The increases in enzyme activities were also accompanied by 3-fold increases in immunoreactive P450IIE protein and its mRNA. In contrast, no differences were observed for the catalytic activities of N-alkoxyresorufin dealkylases or the amounts of immunoreactive P450IA and P450IIC, indicating a specific induction of P450IIE by high fat feeding. Furthermore, the increases in the levels of P450IIE mRNA correlated positively (r = 0.73) with plasma concentrations of acetoacetate and beta-hydroxybutyrate but not with that of acetone, which induces P450IIE without changing its mRNA level. Our data thus indicated that P450IIE induction during the ketosis of a high fat feeding appears to be due to pretranslational activation and that is similar to the induction mechanism of fasted and diabetic animals.

Research paper thumbnail of Phase I clinical evaluation of seasonal influenza hemagglutinin (HA) DNA vaccine prime followed by trivalent influenza inactivated vaccine (IIV3) boost

Contemporary Clinical Trials, 2015

Annual influenza vaccination reduces the risks of influenza when the vaccines are well matched to... more Annual influenza vaccination reduces the risks of influenza when the vaccines are well matched to circulating strains, but development of an approach that induces broader and more durable immune responses would be beneficial. We conducted two companion Phase 1 studies, VRC 307 and VRC 309, over sequential seasons (2008-2009 and 2009-2010) in which only the influenza B strain component of the vaccines differed. Objectives were safety and immunogenicity of prime-boost vaccination schedules. A schedule of DNA vaccine encoding for seasonal influenza hemagglutinins (HA) prime followed by seasonal trivalent influenza inactivated vaccine (IIV3) boost (HA DNA-IIV3) was compared to placebo (PBS)-IIV3 or IIV3-IIV3. Cumulatively, 111 adults were randomized to HA DNA-IIV3 (n = 66), PBS-IIV3 (n = 25) or IIV3-IIV3 (n = 20). Safety was assessed by clinical observations, laboratory parameters and 7-day solicited reactogenicity. The seasonal HA DNA prime-IIV3 boost regimen was evaluated as safe and well tolerated. There were no serious adverse events. The local and systemic reactogenicity for HA DNA, IIV and placebo were reported predominantly as none or mild within the first 5 days postvaccination. There was no significant difference in immunogenicity detected between the treatment groups as evaluated by hemagglutination inhibition (HAI) assay. The studies demonstrated the safety and immunogenicity of seasonal HA DNA-IIV3 regimen, but the 3-4 week prime-boost interval was suboptimal for improving influenza-specific immune responses. This is consistent with observations in avian H5 DNA vaccine prime-boost studies in which a long *

Research paper thumbnail of Safety and tolerability of AAV8 delivery of a broadly neutralizing antibody in adults living with HIV: a phase 1, dose-escalation trial

Nature Medicine

Adeno-associated viral vector-mediated transfer of DNA coding for broadly neutralizing anti-HIV a... more Adeno-associated viral vector-mediated transfer of DNA coding for broadly neutralizing anti-HIV antibodies (bnAbs) offers an alternative to attempting to induce protection by vaccination or by repeated infusions of bnAbs. In this study, we administered a recombinant bicistronic adeno-associated virus (AAV8) vector coding for both the light and heavy chains of the potent broadly neutralizing HIV-1 antibody VRC07 (AAV8-VRC07) to eight adults living with HIV. All participants remained on effective anti-retroviral therapy (viral load (VL) <50 copies per milliliter) throughout this phase 1, dose-escalation clinical trial ( NCT03374202 ). AAV8-VRC07 was given at doses of 5 × 1010, 5 × 1011 and 2.5 × 1012 vector genomes per kilogram by intramuscular (IM) injection. Primary endpoints of this study were to assess the safety and tolerability of AAV8-VRC07; to determine the pharmacokinetics and immunogenicity of in vivo VRC07 production; and to describe the immune response directed against AAV8-VRC07 vector and its products. Secondary endpoints were to assess the clinical effects of AAV8-VRC07 on CD4 T cell count and VL and to assess the persistence of VRC07 produced in participants. In this cohort, IM injection of AAV8-VRC07 was safe and well tolerated. No clinically significant change in CD4 T cell count or VL occurred during the 1-3 years of follow-up reported here. In participants who received AAV8-VRC07, concentrations of VRC07 were increased 6 weeks (P = 0.008) and 52 weeks (P = 0.016) after IM injection of the product. All eight individuals produced measurable amounts of serum VRC07, with maximal VRC07 concentrations >1 µg ml-1 in three individuals. In four individuals, VRC07 serum concentrations remained stable near maximal concentration for up to 3 years of follow-up. In exploratory analyses, neutralizing activity of in vivo produced VRC07 was similar to that of in vitro produced VRC07. Three of eight participants showed a non-idiotypic anti-drug antibody (ADA) response directed against the Fab portion of VRC07. This ADA response appeared to decrease the production of serum VRC07 in two of these three participants. These data represent a proof of concept that adeno-associated viral vectors can durably produce biologically active, difficult-to-induce bnAbs in vivo, which could add valuable new tools to the fight against infectious diseases.

Research paper thumbnail of Safety and immunogenicity of a ferritin nanoparticle H2 influenza vaccine in healthy adults: a phase 1 trial

Nature Medicine, 2022

easonal influenza epidemics and the threat of pandemic influenza outbreaks are perennial global p... more easonal influenza epidemics and the threat of pandemic influenza outbreaks are perennial global public health challenges. In an average year, seasonal influenza epidemics cause 3-5 million cases of severe illness and 300,000-650,000 deaths worldwide 1,2 , with pandemics capable of much greater morbidity and mortality 3. Vaccination is an essential tool for seasonal influenza control; however, currently licensed influenza vaccines require annual reformulation and immunization due to their specificity for the variable circulating strains 4. Influenza viruses are diverse, with many antigenically unique strains of each seasonal subtype circulating simultaneously in the human population. Even when the circulating viruses are well matched to the strains within the vaccine, the effectiveness of seasonal vaccines typically ranges from 40% to 60%, likely due to the lack of adjuvants in the vaccines and the pre-existing immunity to the seasonal subtypes in the population 1. Influenza A hemagglutinin (HA) glycoproteins, which mediate binding and entry of host cells, are phylogenetically categorized into two groups: group 1 (for example, H1, H2, H5, H6 and H9) and group 2 (for example, H3, H7 and H10). The H1 and H3 subtypes are currently responsible for the seasonal epidemics, whereas several other group 1 and group 2 subtypes display pandemic potential 5. Currently licensed seasonal influenza vaccines predominantly elicit

Research paper thumbnail of Autocrine Production of b-Chemokines Protects CMV-Specific CD4+ T Cells from HIV Infection

Induction of a functional subset of HIV-specific CD4+ T cells that is resistant to HIV infection ... more Induction of a functional subset of HIV-specific CD4+ T cells that is resistant to HIV infection could enhance immune protection and decrease the rate of HIV disease progression. CMV-specific CD4+ T cells, which are less frequently infected than HIV-specific CD4+ T cells, are a model for such an effect. To determine the mechanism of this protection, we compared the functional response of HIV gag-specific and CMV pp65-specific CD4+ T cells in individuals co-infected with CMV and HIV. We found that CMV-specific CD4+ T cells rapidly up-regulated production of MIP-1a and MIP-1b mRNA, resulting in a rapid increase in production of MIP-1a and MIP-1b after cognate antigen stimulation. Production of b-chemokines was associated with maturational phenotype and was rarely seen in HIV-specific CD4+ T cells. To test whether production of b-chemokines by CD4+ T cells lowers their susceptibility to HIV infection, we measured cell-associated Gag DNA to assess the in vivo infection history of CMV-sp...

Research paper thumbnail of The metabolism of acetone in rat

Journal of Biological Chemistry, 1984

Intraperitoneal injection of 5 pmol of acetone/g, body weight, into 3 rats previously fed 1% acet... more Intraperitoneal injection of 5 pmol of acetone/g, body weight, into 3 rats previously fed 1% acetone (v/v) in their drinking water resulted in the appearance in blood serum of 16 k 2 nmol of 1,2-propanediol/ml and 8 f 1 nmol of 2,3-butanediol/ml. N o detectable 1,2propanediol or 2,3-butanediol was found in the serum of animals after acetone or saline injection without prior addition of acetone to drinking water or in the serum of animals injected with saline after having been maintained on drinking water containing 1% acetone. These data suggest that acetone both acts to induce a critical enzyme or enzymes and serves as a precursor for the production of 1,2-propanediol. It is also clear from these data that chronic acetone feeding plays a role in 2,3-butanediol production in the rat. Microsomes isolated from the liver of animals maintained on drinking water supplemented with 1% acetone contained two previously unreported enzymatic activities, acetone monooxygenase which converts acetone to acetol and acetol monooxygenase which converts acetol to methylglyoxal. Both activities require O2 and NADPH. Prior treatment with acetone increased serum D-lactate from 9 nmol/ml f 9 nmol/ml in control animals to 77 f 36 nmol/ml in acetone-fed animals after injection with 5 pmol of acetone/g, body weight. This is consistent with methylglyoxal being a by-product of acetone metabolism. Two pathways for the conversion of acetone to glucose are proposed, the methylglyoxal and the propanediol pathways. The methylglyoxal pathway is responsible for the conversion of acetone to acetol, acetol to methylglyoxal, and the subsequent conversion of methylglyoxal to glucose. The propanediol pathway involves the conversion of acetol to ~-1,2-propanediol by an as yet unknown process. ~-1,2-Propanediol is converted to L-lactaldehyde by alcohol dehydrogenase, and L-lactaldehyde is converted to L-lactic acid by aldehyde dehydrogenase. Expression of these metabolic pathways in rat appears to be dependent on the induction of acetone monooxygenase and acetol monooxygenase by acetone. Recent work by Reichard et al. (1) indicates that acetone may be a significant gluconeogenic precursor in fasting humans. Reichard et al. (1) showed that blood acetone, generated by the decarboxylation of acetoacetate, can reach levels of 1.6 mM in the blood of fasted humans. Their data suggest that up to two-thirds of the circulating blood acetone may be converted to glucose. This glucose production could account for 10% of the gluconeogenic demands of humans fasted 21 days (1) and suggests that acetone is an intermediate in the * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Research paper thumbnail of The interdependence of glycolytic and pentose cycle intermediates in ad libitum fed rats

Journal of Biological Chemistry, 1986

Equilibrium constants for reactions catalyzed by ribulose-5-phosphate 3-epimerase, [sigma xylulos... more Equilibrium constants for reactions catalyzed by ribulose-5-phosphate 3-epimerase, [sigma xylulose-5-P]/[sigma ribulose-5-P] = 1.82, ribose-5-phosphate isomerase, [sigma Rib-5-P]/[sigma ribulose-5-P] = 1.20, transaldolase, [sigma erythrose-4-P] [sigma Fru-6-P]/[sigma sedoheptulose-7-P] [sigma glyceraldehyde 3-P] = 0.37, and transketolase, [sigma Fru-6-P] [sigma glyceraldehyde 3-P]/[sigma erythrose-4-P] [sigma xylulose-5-P] = 29.7 and [sigma Rib-5-P] [sigma xylulose-5-P]/[sigma sedoheptulose-7-P] [sigma glyceraldehyde 3-P] = 0.48, were redetermined under physiological conditions. The equilibrium constant for the combined glucose-6-P dehydrogenase and 6-phosphoglucono-gamma-lactonase reaction, [6-phosphogluconate3-] [NADPH] [H+]2/[Glc-6-P2-] [NADP+], was found to be at least 1 X 10(-9). Using these redetermined equilibrium constants, calculated values of pentose cycle intermediates, based on near equilibrium assumptions and the tissue content of Fru-6-P and glyceraldehyde 3-P, were found to be in good agreement with measured values for male Wistar rats injected with saline, 20 mumol/g pyruvate, 20 mumol/g gluconate, and 20 mumol/g ribose. Measured and calculated values for pentose cycle intermediates in saline injected animals were ribulose-5-P; 3.8 +/- 0.4 and 2.4 +/- 0.1 nmol/g; xylulose-5-P, 5.9 +/- 0.6 nmol/g and 4.3 +/- 0.2 nmol/g; sedoheptulose-7-P, 41.5 +/- 2.4 and 37.6 +/- 2.9 nmol/g; and combined sedopheptulose-7-P and Rib-5-P, 43.0 +/- 2.8 nmol/g and 40.5 +/- 3.0 nmol/g; liver content of erythrose-4-P was less than the detection limits of the assay, 2 nmol/g. Calculated erythrose-4-P was 0.23 +/- 0.01 nmol/g. Liver content of 6-phosphogluconate was 8.5 +/- 0.7 nmol/g. The free cytosolic [NADP+]/[NADPH] ratio calculated from the 6-phosphogluconate dehydrogenase redox couple, 0.0030 +/- 0.0002, was also in good agreement with that calculated from the malic enzyme redox couple, 0.0051 +/- 0.0007, and the isocitrate dehydrogenase redox couple, 0.0066 +/- 0.0008. These data indicate the interdependence of the liver content of glycolytic intermediates and pentose cycle intermediates in ad libitum fed rats.

Research paper thumbnail of Principles Governing Establishment versus Collapse of HIV-1 Cellular Spread

Cell Host & Microbe, 2019

Highlights d Transition from latency to exponential HIV growth is covert, rare, and stochastic d ... more Highlights d Transition from latency to exponential HIV growth is covert, rare, and stochastic d After latency disruption, the initial HIV release amount is highly variable d If the initial virus release exceeds a critical threshold, exponential spread ensues d Coupling experimental and computational approaches can define the origin of HIV rebound

Research paper thumbnail of Purification, characterization and initial rate kinetics of acyl-phosphate-hexose phosphotransferase from Aerobacter aerogenes

Research paper thumbnail of Hydroxylation of acetone by ethanol-inducible P-450 isozyme 3a

Federation Proceedings, 1985

Research paper thumbnail of Early Depletion of<i>Mycobacterium tuberculosis</i>–Specific T Helper 1 Cell Responses after HIV‐1 Infection

The Journal of Infectious Diseases, Dec 1, 2008

Background. The acid-fast bacillus Mycobacterium tuberculosis is often the first manifestation of... more Background. The acid-fast bacillus Mycobacterium tuberculosis is often the first manifestation of acquired immunodeficiency syndrome in patients infected with human immunodeficiency virus (HIV). This study was conducted to better understand the mechanism underlying M. tuberculosis-specific pathogenicity early after onset of HIV infection. Methods. M. tuberculosis-specific T helper 1 (Th1) cells were studied in HIV negative (n ϭ 114) and chronically HIV infected (n ϭ 68) Tanzanian subjects by using early secreted antigenic target 6 (ESAT6) protein or tuberculin (purified protein derivative) with interferon-␥ ELISPOT and intracellular cytokine staining. In a longitudinal study, the effect of acute HIV infection on M. tuberculosis-specific Th1 cells was determined by polychromatic flow cytometric analysis in 5 subjects with latent M. tuberculosis infection who became infected with HIV. Results. In tuberculosis (TB)-asymptomatic subjects (i.e., subjects with unknown TB status who did not show clinical signs suggestive of TB), chronic HIV infection was associated with a decreased percentage of subjects with detectable M. tuberculosis-specific Th1 cells (P Ͻ .001), a decrease which was not observed among subjects with active TB. Acute HIV infection induced a rapid depletion of M. tuberculosis-specific Th1 cells in 4 subjects remained TB asymptomatic, whereas the population of these cells remained stable in subjects who remained HIV negative (P Ͻ .01). Conclusions. Taken together, these data suggest a mechanism of rapid M. tuberculosis-specific Th1 cell depletion that may contribute to the early onset of TB in individuals with latent M. tuberculosis infection who become HIV infected.

Research paper thumbnail of TCR recognition is a mediator in the control of HIV infection by CD8 T cells

Journal of Immunology, May 1, 2017

Research paper thumbnail of Trispecific antibody targeting HIV-1 and T cells activates and eliminates latently-infected cells in HIV/SHIV infections

Nature Communications

Agents that can simultaneously activate latent HIV, increase immune activation and enhance the ki... more Agents that can simultaneously activate latent HIV, increase immune activation and enhance the killing of latently-infected cells represent promising approaches for HIV cure. Here, we develop and evaluate a trispecific antibody (Ab), N6/αCD3-αCD28, that targets three independent proteins: (1) the HIV envelope via the broadly reactive CD4-binding site Ab, N6; (2) the T cell antigen CD3; and (3) the co-stimulatory molecule CD28. We find that the trispecific significantly increases antigen-specific T-cell activation and cytokine release in both CD4+ and CD8+ T cells. Co-culturing CD4+ with autologous CD8+ T cells from ART-suppressed HIV+ donors with N6/αCD3-αCD28, results in activation of latently-infected cells and their elimination by activated CD8+ T cells. This trispecific antibody mediates CD4+ and CD8+ T-cell activation in non-human primates and is well tolerated in vivo. This HIV-directed antibody therefore merits further development as a potential intervention for the eradicati...

Research paper thumbnail of An influenza hemagglutinin stem nanoparticle vaccine induces cross-group 1 neutralizing antibodies in healthy adults

Science Translational Medicine

Influenza vaccines could be improved by platforms inducing cross-reactive immunity. Immunodominan... more Influenza vaccines could be improved by platforms inducing cross-reactive immunity. Immunodominance of the influenza hemagglutinin (HA) head in currently licensed vaccines impedes induction of cross-reactive neutralizing stem-directed antibodies. A vaccine without the variable HA head domain has the potential to focus the immune response on the conserved HA stem. This first-in-human dose-escalation open-label phase 1 clinical trial (NCT03814720) tested an HA stabilized stem ferritin nanoparticle vaccine (H1ssF) based on the H1 HA stem of A/New Caledonia/20/1999. Fifty-two healthy adults aged 18 to 70 years old enrolled to receive either 20 μg of H1ssF once ( n = 5) or 60 μg of H1ssF twice ( n = 47) with a prime-boost interval of 16 weeks. Thirty-five (74%) 60-μg dose participants received the boost, whereas 11 (23%) boost vaccinations were missed because of public health restrictions in the early stages of the COVID-19 pandemic. The primary objective of this trial was to evaluate ...

Research paper thumbnail of Potent anti-viral activity of a trispecific HIV neutralizing antibody in SHIV-infected monkeys

Cell Reports

Broadly neutralizing antibodies (bNAbs) represent an alternative to drug therapy for the treatmen... more Broadly neutralizing antibodies (bNAbs) represent an alternative to drug therapy for the treatment of HIV-1 infection. Immunotherapy with single bNAbs often leads to emergence of escape variants, suggesting a potential benefit of combination bNAb therapy. Here, a trispecific bNAb reduces viremia 100- to 1000-fold in viremic SHIV-infected macaques. After treatment discontinuation, viremia rebounds transiently and returns to low levels, through CD8-mediated immune control. These viruses remain sensitive to the trispecific antibody, despite loss of sensitivity to one of the parental bNAbs. Similarly, the trispecific bNAb suppresses the emergence of resistance in viruses derived from HIV-1-infected subjects, in contrast to parental bNAbs. Trispecific HIV-1 neutralizing antibodies, therefore, mediate potent antiviral activity in vivo and may minimize the potential for immune escape.

Research paper thumbnail of Application of B cell immortalization for the isolation of antibodies and B cell clones from vaccine and infection settings

The isolation and characterization of neutralizing antibodies from infection and vaccine settings... more The isolation and characterization of neutralizing antibodies from infection and vaccine settings will inform future vaccine design, and methodologies that streamline the isolation of antibodies and the generation of B cell clones are of great interest. Retroviral transduction to express Bcl-6 and Bcl-xL in primary B cells has been shown to promote long-term B cell survival and antibody secretion in vitro, and can be used to isolate antibodies from memory B cells. The application of this methodology to B cell subsets from tissues and to B cells from individuals with chronic infection has not been extensively characterized. Here, we characterize Bcl-6/Bcl-xL B cell immortalization across multiple tissue types and B cell subsets in healthy and HIV-1 infected individuals, as well as individuals recovering from malaria. In HIV-1- and malaria-uninfected donors, naïve and memory B cell subsets from PBMC and tonsil tissue transformed with similar efficiencies, and displayed similar charact...

Research paper thumbnail of Th1/Th17 cell induction following vaccination with the novel tuberculosis vaccine MVA85A and their role in Mycobacterium tuberculosis challengeCD4 T cell response to MVA85A vaccination against Mycobacterium tuberculosis in healthy HIV-infected adults SO IMMUNOLOGY CT Annual Congress of the Britis...

Research paper thumbnail of CD4 T cell response to MVA85A vaccination against Mycobacterium tuberculosis in healthy HIV-infected adults

Research paper thumbnail of Sustained HIV release by single persisting CD4 + T cells during latency disruption

Journal of Virus Eradication, 2015

cells had returned to resting. Second, cell proliferation of acutely infected cultures was tracke... more cells had returned to resting. Second, cell proliferation of acutely infected cultures was tracked using CFSE dye and cell subsets that had undergone varying degrees of proliferation were isolated by FACS, at the end of culture. Each recovered cell subset was analyzed for the quantity of integrated HIV DNA and replication-competent virus. Results: Cells exposed to HIV, prior to stimulation, contained the highest levels of integrated provirus and replication competent virus after returning to quiescence. Cells infected, during the height of cell proliferation, retained the least HIV. Cells that did not divide or divided only a few times contained higher levels of integrated and replicationcompetent HIV than did cells, which had divided many times. Conclusions: HIV latency is established very early within a heterogeneous population of CD4 T cells, which are undergoing varying degrees of cell activation in the presence of infectious virus. Minimally activated cells within this milieu are the most likely to develop persistent latent infection. These observations have important implications for the study of latency, and may aid in the design of strategies to purge this reservoir.

Research paper thumbnail of Uncovering mechanisms of HIV persistence in HIV controllers by HIV sequence analysis in CD4 T cell subsets

Journal of Virus Eradication, 2015

or more of these latency-reversing agents (LRAs) will be combined with a vaccine capable of elici... more or more of these latency-reversing agents (LRAs) will be combined with a vaccine capable of eliciting cytotoxic T cells to clear reactivated cells. Therefore, we studied the effects of these drugs on T cells. Methods: We examined the in vitro impact of clinically-relevant exposures to putative LRAs on T cell activation and function. Histone deacetylase inhibitors (HDACi) vorinostat (VOR), panobinostat (PAN) and romidepsin (ROMI) were compared with protein kinase C modulators ingenol 3,20 dibenzoate (ING), prostratin (PRO) and bryostatin-1 (BRYO). Results: No effect of VOR was observed on T cell activation, whereas PAN and ROMI consistently increased expression of CD69 on CD4 and CD8 T cells. PKC modulators all induced increased expression of CD69 and MHC I on CD4 and CD8 T cells, which for CD69 was sustained over 72 hours. VOR and ROMI did not impact ex vivo antigen-specific CD8 T cell responses as measured by degranulation and cytokine production six hours after stimulation. PAN, however, produced a small but significant decrease in the frequency of antigen-specific CD8 T cell responses. PKC modulators all induced non-specific cytokine production, an effect that was more marked in HIV seropositive than in seronegative individuals, but did not significantly affect antigen-specific responses. Interestingly, ROM, PRO and BRYO inhibited antigen-dependent proliferation over a 5 day in vitro culture in both seropositive and seronegative participants. PRO and BRYO were toxic in these short term culture assays. Conclusions: Even within the same class, LRAs vary in their effect on the phenotypic profile and functional capacity of T cells. Moreover, effects on T cell function may not be detectable until several days after dosing. These differences should be considered for dosing, designing and testing of HIV latency clearance strategies.

Research paper thumbnail of Pretranslational activation of cytochrome P450IIE during ketosis induced by a high fat diet

PubMed, Mar 1, 1992

Ethanol-inducible cytochrome P450 (P450IIE) is reported to be induced by ketosis. In the present ... more Ethanol-inducible cytochrome P450 (P450IIE) is reported to be induced by ketosis. In the present study, the effects of a high fat diet on P450IIE induction and the relationship between ketone body concentration and P450IIE induction were studied by the following: 1) measurement of the activity of aniline hydroxylase, 2) immunoblot analysis for P450IIE protein, and 3) Northern blot analysis for P450IIE mRNA. The enzyme activities (aniline hydroxylase) in hepatic and renal microsomes were elevated about 2-3-fold by feeding with a high fat diet for 3 days. The increases in enzyme activities were also accompanied by 3-fold increases in immunoreactive P450IIE protein and its mRNA. In contrast, no differences were observed for the catalytic activities of N-alkoxyresorufin dealkylases or the amounts of immunoreactive P450IA and P450IIC, indicating a specific induction of P450IIE by high fat feeding. Furthermore, the increases in the levels of P450IIE mRNA correlated positively (r = 0.73) with plasma concentrations of acetoacetate and beta-hydroxybutyrate but not with that of acetone, which induces P450IIE without changing its mRNA level. Our data thus indicated that P450IIE induction during the ketosis of a high fat feeding appears to be due to pretranslational activation and that is similar to the induction mechanism of fasted and diabetic animals.

Research paper thumbnail of Phase I clinical evaluation of seasonal influenza hemagglutinin (HA) DNA vaccine prime followed by trivalent influenza inactivated vaccine (IIV3) boost

Contemporary Clinical Trials, 2015

Annual influenza vaccination reduces the risks of influenza when the vaccines are well matched to... more Annual influenza vaccination reduces the risks of influenza when the vaccines are well matched to circulating strains, but development of an approach that induces broader and more durable immune responses would be beneficial. We conducted two companion Phase 1 studies, VRC 307 and VRC 309, over sequential seasons (2008-2009 and 2009-2010) in which only the influenza B strain component of the vaccines differed. Objectives were safety and immunogenicity of prime-boost vaccination schedules. A schedule of DNA vaccine encoding for seasonal influenza hemagglutinins (HA) prime followed by seasonal trivalent influenza inactivated vaccine (IIV3) boost (HA DNA-IIV3) was compared to placebo (PBS)-IIV3 or IIV3-IIV3. Cumulatively, 111 adults were randomized to HA DNA-IIV3 (n = 66), PBS-IIV3 (n = 25) or IIV3-IIV3 (n = 20). Safety was assessed by clinical observations, laboratory parameters and 7-day solicited reactogenicity. The seasonal HA DNA prime-IIV3 boost regimen was evaluated as safe and well tolerated. There were no serious adverse events. The local and systemic reactogenicity for HA DNA, IIV and placebo were reported predominantly as none or mild within the first 5 days postvaccination. There was no significant difference in immunogenicity detected between the treatment groups as evaluated by hemagglutination inhibition (HAI) assay. The studies demonstrated the safety and immunogenicity of seasonal HA DNA-IIV3 regimen, but the 3-4 week prime-boost interval was suboptimal for improving influenza-specific immune responses. This is consistent with observations in avian H5 DNA vaccine prime-boost studies in which a long *

Research paper thumbnail of Safety and tolerability of AAV8 delivery of a broadly neutralizing antibody in adults living with HIV: a phase 1, dose-escalation trial

Nature Medicine

Adeno-associated viral vector-mediated transfer of DNA coding for broadly neutralizing anti-HIV a... more Adeno-associated viral vector-mediated transfer of DNA coding for broadly neutralizing anti-HIV antibodies (bnAbs) offers an alternative to attempting to induce protection by vaccination or by repeated infusions of bnAbs. In this study, we administered a recombinant bicistronic adeno-associated virus (AAV8) vector coding for both the light and heavy chains of the potent broadly neutralizing HIV-1 antibody VRC07 (AAV8-VRC07) to eight adults living with HIV. All participants remained on effective anti-retroviral therapy (viral load (VL) <50 copies per milliliter) throughout this phase 1, dose-escalation clinical trial ( NCT03374202 ). AAV8-VRC07 was given at doses of 5 × 1010, 5 × 1011 and 2.5 × 1012 vector genomes per kilogram by intramuscular (IM) injection. Primary endpoints of this study were to assess the safety and tolerability of AAV8-VRC07; to determine the pharmacokinetics and immunogenicity of in vivo VRC07 production; and to describe the immune response directed against AAV8-VRC07 vector and its products. Secondary endpoints were to assess the clinical effects of AAV8-VRC07 on CD4 T cell count and VL and to assess the persistence of VRC07 produced in participants. In this cohort, IM injection of AAV8-VRC07 was safe and well tolerated. No clinically significant change in CD4 T cell count or VL occurred during the 1-3 years of follow-up reported here. In participants who received AAV8-VRC07, concentrations of VRC07 were increased 6 weeks (P = 0.008) and 52 weeks (P = 0.016) after IM injection of the product. All eight individuals produced measurable amounts of serum VRC07, with maximal VRC07 concentrations >1 µg ml-1 in three individuals. In four individuals, VRC07 serum concentrations remained stable near maximal concentration for up to 3 years of follow-up. In exploratory analyses, neutralizing activity of in vivo produced VRC07 was similar to that of in vitro produced VRC07. Three of eight participants showed a non-idiotypic anti-drug antibody (ADA) response directed against the Fab portion of VRC07. This ADA response appeared to decrease the production of serum VRC07 in two of these three participants. These data represent a proof of concept that adeno-associated viral vectors can durably produce biologically active, difficult-to-induce bnAbs in vivo, which could add valuable new tools to the fight against infectious diseases.

Research paper thumbnail of Safety and immunogenicity of a ferritin nanoparticle H2 influenza vaccine in healthy adults: a phase 1 trial

Nature Medicine, 2022

easonal influenza epidemics and the threat of pandemic influenza outbreaks are perennial global p... more easonal influenza epidemics and the threat of pandemic influenza outbreaks are perennial global public health challenges. In an average year, seasonal influenza epidemics cause 3-5 million cases of severe illness and 300,000-650,000 deaths worldwide 1,2 , with pandemics capable of much greater morbidity and mortality 3. Vaccination is an essential tool for seasonal influenza control; however, currently licensed influenza vaccines require annual reformulation and immunization due to their specificity for the variable circulating strains 4. Influenza viruses are diverse, with many antigenically unique strains of each seasonal subtype circulating simultaneously in the human population. Even when the circulating viruses are well matched to the strains within the vaccine, the effectiveness of seasonal vaccines typically ranges from 40% to 60%, likely due to the lack of adjuvants in the vaccines and the pre-existing immunity to the seasonal subtypes in the population 1. Influenza A hemagglutinin (HA) glycoproteins, which mediate binding and entry of host cells, are phylogenetically categorized into two groups: group 1 (for example, H1, H2, H5, H6 and H9) and group 2 (for example, H3, H7 and H10). The H1 and H3 subtypes are currently responsible for the seasonal epidemics, whereas several other group 1 and group 2 subtypes display pandemic potential 5. Currently licensed seasonal influenza vaccines predominantly elicit

Research paper thumbnail of Autocrine Production of b-Chemokines Protects CMV-Specific CD4+ T Cells from HIV Infection

Induction of a functional subset of HIV-specific CD4+ T cells that is resistant to HIV infection ... more Induction of a functional subset of HIV-specific CD4+ T cells that is resistant to HIV infection could enhance immune protection and decrease the rate of HIV disease progression. CMV-specific CD4+ T cells, which are less frequently infected than HIV-specific CD4+ T cells, are a model for such an effect. To determine the mechanism of this protection, we compared the functional response of HIV gag-specific and CMV pp65-specific CD4+ T cells in individuals co-infected with CMV and HIV. We found that CMV-specific CD4+ T cells rapidly up-regulated production of MIP-1a and MIP-1b mRNA, resulting in a rapid increase in production of MIP-1a and MIP-1b after cognate antigen stimulation. Production of b-chemokines was associated with maturational phenotype and was rarely seen in HIV-specific CD4+ T cells. To test whether production of b-chemokines by CD4+ T cells lowers their susceptibility to HIV infection, we measured cell-associated Gag DNA to assess the in vivo infection history of CMV-sp...

Research paper thumbnail of The metabolism of acetone in rat

Journal of Biological Chemistry, 1984

Intraperitoneal injection of 5 pmol of acetone/g, body weight, into 3 rats previously fed 1% acet... more Intraperitoneal injection of 5 pmol of acetone/g, body weight, into 3 rats previously fed 1% acetone (v/v) in their drinking water resulted in the appearance in blood serum of 16 k 2 nmol of 1,2-propanediol/ml and 8 f 1 nmol of 2,3-butanediol/ml. N o detectable 1,2propanediol or 2,3-butanediol was found in the serum of animals after acetone or saline injection without prior addition of acetone to drinking water or in the serum of animals injected with saline after having been maintained on drinking water containing 1% acetone. These data suggest that acetone both acts to induce a critical enzyme or enzymes and serves as a precursor for the production of 1,2-propanediol. It is also clear from these data that chronic acetone feeding plays a role in 2,3-butanediol production in the rat. Microsomes isolated from the liver of animals maintained on drinking water supplemented with 1% acetone contained two previously unreported enzymatic activities, acetone monooxygenase which converts acetone to acetol and acetol monooxygenase which converts acetol to methylglyoxal. Both activities require O2 and NADPH. Prior treatment with acetone increased serum D-lactate from 9 nmol/ml f 9 nmol/ml in control animals to 77 f 36 nmol/ml in acetone-fed animals after injection with 5 pmol of acetone/g, body weight. This is consistent with methylglyoxal being a by-product of acetone metabolism. Two pathways for the conversion of acetone to glucose are proposed, the methylglyoxal and the propanediol pathways. The methylglyoxal pathway is responsible for the conversion of acetone to acetol, acetol to methylglyoxal, and the subsequent conversion of methylglyoxal to glucose. The propanediol pathway involves the conversion of acetol to ~-1,2-propanediol by an as yet unknown process. ~-1,2-Propanediol is converted to L-lactaldehyde by alcohol dehydrogenase, and L-lactaldehyde is converted to L-lactic acid by aldehyde dehydrogenase. Expression of these metabolic pathways in rat appears to be dependent on the induction of acetone monooxygenase and acetol monooxygenase by acetone. Recent work by Reichard et al. (1) indicates that acetone may be a significant gluconeogenic precursor in fasting humans. Reichard et al. (1) showed that blood acetone, generated by the decarboxylation of acetoacetate, can reach levels of 1.6 mM in the blood of fasted humans. Their data suggest that up to two-thirds of the circulating blood acetone may be converted to glucose. This glucose production could account for 10% of the gluconeogenic demands of humans fasted 21 days (1) and suggests that acetone is an intermediate in the * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Research paper thumbnail of The interdependence of glycolytic and pentose cycle intermediates in ad libitum fed rats

Journal of Biological Chemistry, 1986

Equilibrium constants for reactions catalyzed by ribulose-5-phosphate 3-epimerase, [sigma xylulos... more Equilibrium constants for reactions catalyzed by ribulose-5-phosphate 3-epimerase, [sigma xylulose-5-P]/[sigma ribulose-5-P] = 1.82, ribose-5-phosphate isomerase, [sigma Rib-5-P]/[sigma ribulose-5-P] = 1.20, transaldolase, [sigma erythrose-4-P] [sigma Fru-6-P]/[sigma sedoheptulose-7-P] [sigma glyceraldehyde 3-P] = 0.37, and transketolase, [sigma Fru-6-P] [sigma glyceraldehyde 3-P]/[sigma erythrose-4-P] [sigma xylulose-5-P] = 29.7 and [sigma Rib-5-P] [sigma xylulose-5-P]/[sigma sedoheptulose-7-P] [sigma glyceraldehyde 3-P] = 0.48, were redetermined under physiological conditions. The equilibrium constant for the combined glucose-6-P dehydrogenase and 6-phosphoglucono-gamma-lactonase reaction, [6-phosphogluconate3-] [NADPH] [H+]2/[Glc-6-P2-] [NADP+], was found to be at least 1 X 10(-9). Using these redetermined equilibrium constants, calculated values of pentose cycle intermediates, based on near equilibrium assumptions and the tissue content of Fru-6-P and glyceraldehyde 3-P, were found to be in good agreement with measured values for male Wistar rats injected with saline, 20 mumol/g pyruvate, 20 mumol/g gluconate, and 20 mumol/g ribose. Measured and calculated values for pentose cycle intermediates in saline injected animals were ribulose-5-P; 3.8 +/- 0.4 and 2.4 +/- 0.1 nmol/g; xylulose-5-P, 5.9 +/- 0.6 nmol/g and 4.3 +/- 0.2 nmol/g; sedoheptulose-7-P, 41.5 +/- 2.4 and 37.6 +/- 2.9 nmol/g; and combined sedopheptulose-7-P and Rib-5-P, 43.0 +/- 2.8 nmol/g and 40.5 +/- 3.0 nmol/g; liver content of erythrose-4-P was less than the detection limits of the assay, 2 nmol/g. Calculated erythrose-4-P was 0.23 +/- 0.01 nmol/g. Liver content of 6-phosphogluconate was 8.5 +/- 0.7 nmol/g. The free cytosolic [NADP+]/[NADPH] ratio calculated from the 6-phosphogluconate dehydrogenase redox couple, 0.0030 +/- 0.0002, was also in good agreement with that calculated from the malic enzyme redox couple, 0.0051 +/- 0.0007, and the isocitrate dehydrogenase redox couple, 0.0066 +/- 0.0008. These data indicate the interdependence of the liver content of glycolytic intermediates and pentose cycle intermediates in ad libitum fed rats.

Research paper thumbnail of Principles Governing Establishment versus Collapse of HIV-1 Cellular Spread

Cell Host & Microbe, 2019

Highlights d Transition from latency to exponential HIV growth is covert, rare, and stochastic d ... more Highlights d Transition from latency to exponential HIV growth is covert, rare, and stochastic d After latency disruption, the initial HIV release amount is highly variable d If the initial virus release exceeds a critical threshold, exponential spread ensues d Coupling experimental and computational approaches can define the origin of HIV rebound