Timing of plasmid cytokine (IL-2/Ig) administration affects HIV-1 vaccine immunogenicity in HIV-seronegative subjects.

BACKGROUND: To investigate the potential immunostimulatory effect of interleukin (IL) 2 as a human immunodeficiency virus type 1 (HIV-1) vaccine adjuvant, we conducted a study of a plasmid coding for a fusion protein of IL-2 and immunoglobulin (IL-2/Ig). METHODS: This phase I trial evaluated an HIV-1 DNA vaccine with the plasmid cytokine adjuvant (IL-2/Ig) in 70 HIV-negative adults. Subjects received placebo (group C), adjuvant alone (group A), vaccine alone (group D), increasing doses of adjuvant concurrent with vaccine (groups T1-T4), or adjuvant given 2 days after vaccine (group T5). RESULTS: No significant differences in adverse events were observed between treatment groups. Cellular immune responses to envelope protein EnvA peptides were detected by interferon (IFN) γ and IL-2 enzyme-linked immunospot (ELISPOT) assays in 50% and 40% of subjects, respectively, in T4, and in 100% and 80% in T5. The median responses for groups T4 and T5, respectively, were 90 and 193 spot-forming cells (SFCs)/106 peripheral blood mononuclear cells (P = .004; T4 vs T5) for the IL-2 ELISPOT assay and 103 and 380 SFCs/106 PBMCs (P = .003; T4 vs T5) for the IFN-γ ELISPOT assay. A trend to more durable cellular immune responses in T5 was observed at 1 year (T5 vs T4/D; P = .07). Higher anti-Env antibody responses were detected with T5 than with T4. CONCLUSIONS: Plasmid IL-2/Ig significantly increased immune responses when administered 2 days after the DNA vaccine, compared with simultaneous administration. These observations have important implications for the development of cytokine augmentation strategies. CLINICAL TRIALS REGISTRATION: NCT00069030.

Phase 1 safety and immunogenicity testing of DNA and recombinant modified vaccinia Ankara vaccines expressing HIV-1 virus-like particles.

BACKGROUND: Recombinant DNA and modified vaccinia virus Ankara (rMVA) vaccines represent a promising approach to an HIV/AIDS vaccine. This Phase 1 clinical trial compared the safety and immunogenicity of a rMVA vaccine administered with and without DNA vaccine priming METHODS: GeoVax pGA2/JS7 DNA (D) and MVA/HIV62 (M) vaccines encode noninfectious virus-like particles. Intramuscular needle injections were used to deliver placebo, 2 doses of DNA followed by 2 doses of rMVA (DDMM), one dose of DNA followed by 2 doses of rMVA (DMM), or 3 doses of rMVA (MMM) to HIV-seronegative participants. RESULTS: Local and systemic symptoms were mild or moderate. Immune response rates for CD4 + and CD8 + T cells were highest in the DDMM group and lowest in the MMM group (77% vs 43% CD4 + and 42% vs 17% CD8 +). In contrast, response rates for Env binding and neutralizing Ab were highest in the MMM group. The DMM group had intermediate response rates. A 1/10th-dose DDMM regimen induced similar T cell but reduced Ab response rates compared with the full-dose DDMM. CONCLUSIONS: MVA62 was well tolerated and elicited different patterns of T cell and Ab responses when administered alone or in combination with the JS7 DNA vaccine.

VASP phosphorylation at serine239 regulates the effects of NO on smooth muscle cell invasion and contraction of collagen.

Nitric oxide triggers cGMP-dependent kinase-mediated phosphorylation of the actin regulator vasodilator-stimulated phosphoprotein (VASP) at residue serine239. The function of this phosphorylation for smooth muscle cell (SMC) adhesion, spreading, matrix contraction, and invasion is not well understood. We reconstituted VASP deficient SMC with wild-type VASP (wt-VASP) or VASP mutants that mimic "locked" serine239 phosphorylation (S239D-VASP) or "blocked" serine239 phosphorylation (S239A-VASP). Collagen gel contraction was reduced in S239D-VASP compared to S239A-VASP and wt-VASP expressing cells and nitric oxide (NO) stimulation decreased gel contraction of wt-VASP reconstituted SMC. Invasion of collagen was enhanced in S239D-VASP and NO-stimulated wild-type SMCs compared to S239A-VASP expressing cells. Expression of S239D-VASP impaired SMC attachment to collagen, reduced the number of membrane protrusions, and caused cell rounding compared to expression of S239A-VASP. Treatment of wt-VASP reconstituted SMCs with NO exerted similar effects as expression of S239D-VASP. As unstimulated cells were spreading on collagen S239A-VASP and wt-VASP localized to actin fibers whereas S239D-VASP was enriched in the cytosol. NO interferes with SMC invasion and contraction of collagen matrices. This requires phosphorylation of VASP on serine239, which reduces VASP binding to actin fibers. These findings support the conclusion that VASP phosphorylation at serine239 regulates cytoskeleton remodeling.

HIV-1 vaccine-induced immunity in the test-of-concept Step Study: a case-cohort analysis.

BACKGROUND: In the Step Study, the MRKAd5 HIV-1 gag/pol/nef vaccine did not reduce plasma viraemia after infection, and HIV-1 incidence was higher in vaccine-treated than in placebo-treated men with pre-existing adenovirus serotype 5 (Ad5) immunity. We assessed vaccine-induced immunity and its potential contributions to infection risk. METHODS: To assess immunogenicity, we characterised HIV-specific T cells ex vivo with validated interferon-gamma ELISPOT and intracellular cytokine staining assays, using a case-cohort design. To establish effects of vaccine and pre-existing Ad5 immunity on infection risk, we undertook flow cytometric studies to measure Ad5-specific T cells and circulating activated (Ki-67+/BcL-2(lo)) CD4+ T cells expressing CCR5. FINDINGS: We detected interferon-gamma-secreting HIV-specific T cells (range 163/10(6) to 686/10(6) peripheral blood mononuclear cells) ex vivo by ELISPOT in 77% (258/354) of people receiving vaccine; 218 of 354 (62%) recognised two to three HIV proteins. We identified HIV-specific CD4+ T cells by intracellular cytokine staining in 58 of 142 (41%) people. In those with reactive CD4+ T cells, the median percentage of CD4+ T cells expressing interleukin 2 was 88%, and the median co-expression of interferon gamma or tumor necrosis factor alpha (TNFalpha), or both, was 72%. We noted HIV-specific CD8+ T cells (range 0.4-1.0%) in 117 of 160 (73%) participants, expressing predominantly either interferon gamma alone or with TNFalpha. Vaccine-induced HIV-specific immunity, including response rate, magnitude, and cytokine profile, did not differ between vaccinated male cases (before infection) and non-cases. Ad5-specific T cells were lower in cases than in non-cases in several subgroup analyses. The percentage of circulating Ki-67+BcL-2(lo)/CCR5+CD4+ T cells did not differ between cases and non-cases. INTERPRETATION: Consistent with previous trials, the MRKAd5 HIV-1 gag/pol/nef vaccine was highly immunogenic for inducing HIV-specific CD8+ T cells. Our findings suggest that future candidate vaccines have to elicit responses that either exceed in magnitude or differ in breadth or function from those recorded in this trial.

Bi-directional drones to strengthen healthcare provision: experiences and lessons from Madagascar, Malawi and Senegal.

Drones are increasingly being used globally for the support of healthcare programmes. Madagascar, Malawi and Senegal are among a group of early adopters piloting the use of bi-directional transport drones for health systems in sub-Saharan Africa. This article presents the experiences as well as the strengths, weaknesses, opportunities and threats (SWOT analysis) of these country projects. Methods for addressing regulatory, feasibility, acceptability, and monitoring and evaluation issues are presented to guide future implementations. Main recommendations for governments, implementers, drone providers and funders include (1) developing more reliable technologies, (2) thorough vetting of drone providers' capabilities during the selection process, (3) using and strengthening local capacity, (4) building in-country markets and businesses to maintain drone operations locally, (5) coordinating efforts among all stakeholders under government leadership, (6) implementing and identifying funding for long-term projects beyond pilots, and (7) evaluating impacts via standardised indicators. Sharing experiences and evidence from ongoing projects is needed to advance the use of drones for healthcare.

MMP-9 regulates both positively and negatively collagen gel contraction: a nonproteolytic function of MMP-9.

OBJECTIVE: Constrictive remodeling accounts for lumen loss in postangioplasty restenosis. Matrix metalloproteinase-9 (MMP-9) has been shown to prevent constrictive remodeling in vivo. To investigate potential mechanisms for this observation, we investigated the role of MMP-9 in smooth muscle cell (SMC)-mediated collagen gel contraction, an in vitro model of constrictive remodeling. METHODS: Fischer rat SMCs were stably transfected with a construct-expressing rat-MMP-9 under the control of a tetracycline (Tet)-off promoter. SMCs were seeded in type I collagen gels (2.4 mg/ml) in the presence or not of tetracycline (1 microg/ml), and gel contraction was defined as the percentage of retraction of the collagen gel. The depletion of MMP-9 was obtained by using siRNA targeting MMP-9 mRNA or a blocking antibody. RESULTS: Gel contraction was significantly reduced at all times when MMP-9 was overexpressed (Tet-) as compared with the control condition (Tet+). However, MMP-9 depletion of control (Tet+) SMCs (using siRNA or antibody) also inhibited gel contraction. To resolve the apparent discrepancy and determine if MMP-9 exerts a dose-dependent biphasic effect on gel contraction, conditioned medium and purified rat-MMP-9 were prepared. Gel contraction was significantly increased by addition of 0.8 ng/ml of MMP-9, while high concentrations of MMP-9 (> or =100 ng/ml) inhibited contraction. The addition of BB94 and TIMP-1 did not alter the inhibitory or stimulatory effect of MMP-9. CONCLUSIONS: Our data suggest that MMP-9, independent of its proteolytic function, has a biphasic effect on SMC-mediated collagen gel contraction. Understanding the different roles of MMP-9 should allow the development of better therapeutic strategies for restenotic vascular disease.

TIMP-2 and PAI-1 mRNA levels are lower in aneurysmal as compared to athero-occlusive abdominal aortas.

OBJECTIVE: Significant alterations of the vascular wall occurs in abdominal aortic aneurysm (AAA) and atherosclerotic occlusive disease (AOD) that ultimately may lead to either vascular rupture or obstruction. These modifications have been ascribed to one or a group of proteases, their inhibitors or to the matrix macromolecules involved in the repair process without considering the extent of the observed variations. METHODS: The mRNA steady-state level of a large spectrum of proteolytic enzymes (matrix metalloproteinases: MMP-1, -2, -3, -8, -9, -11, -12, -13, -14; urokinase plasminogen activator: u-PA), their physiological inhibitors (tissue inhibitors of MMPs: TIMP-1, -2, -3; plasminogen activator inhibitor: PAI-1) and that of structural matrix proteins (collagens type I and III, decorin, elastin, fibrillins 1 and 2) was determined by RT-PCR made quantitative by using a synthetic RNA as internal standard in each reaction mixture. The profile of expression was evaluated in AAA (n=7) and AOD (n=5) and compared to non-diseased abdominal (CAA, n=7) and thoracic aorta (CTA, n=5). RESULTS: The MMPs -8, -9, -12 and -13 mostly associated with inflammatory cells were not or barely detected in CAA and CTA while they were largely and similarly expressed in AAA and AOD. Expression of protease inhibitors or structural proteins were only slightly increased in both pathological conditions with the exception of elastin which was reduced. The main significant difference between AAA and AOD was a lower expression of TIMP-2 and PAI-1 in the aneurysmal lesions. CONCLUSIONS: The remodeling of the aortic wall in AAA and AOD involves gene activation of a large and similar spectrum of proteolytic enzymes while the expression of two physiological inhibitors, TIMP-2 and PAI-1, is significantly lower in AAA compared to AOD. The repair process in the aneurysmal disease seems similar to that of the occlusive disease.

Optimization and qualification of a multiplex bead array to assess cytokine and chemokine production by vaccine-specific cells.

The magnitude and functional phenotype (e.g. proliferation, immune stimulation) of vaccine-induced T-cell responses are likely to be critical in defining responses that can control pathogenic challenge. Current multi-parameter flow cytometric techniques may not be sufficient to measure all of these different functions, since characterizing T-cell responses by flow cytometry is presently limited to concurrent measurement of at most 10 cytokines/chemokines. Here, we describe extensive studies conducted using standardized GCLP procedures to optimize and qualitatively/quantitatively qualify a multiplex bead array (MBA) performed on supernatant collected from stimulated peripheral blood mononuclear cells (PBMC) to assess 12 cytokines and chemokines of interest. Our optimized MBA shows good precision (intra-assay, inter-day, inter-technician; coefficients of variation <30%) and linearity for most of the analytes studied. We also developed positivity criteria that allow us to define a response as positive or negative with a high degree of confidence. In conclusion, we provide a detailed description of the qualification of an MBA, which permits quantitative and qualitative evaluation of vaccine-induced immunogenicity and analysis of immune correlates of protection. This assay provides an excellent complement to the existing repertoire of assays for assessing immunogenicity in HIV vaccine clinical trials.

Human adenovirus-specific T cells modulate HIV-specific T cell responses to an Ad5-vectored HIV-1 vaccine.

Recombinant viruses hold promise as vectors for vaccines to prevent infectious diseases with significant global health impacts. One of their major limitations is that preexisting anti-vector neutralizing antibodies can reduce T cell responses to the insert antigens; however, the impact of vector-specific cellular immunity on subsequent insert-specific T cell responses has not been assessed in humans. Here, we have identified and compared adenovirus-specific and HIV-specific T cell responses in subjects participating in two HIV-1 vaccine trials using a vaccine vectored by adenovirus serotype 5 (Ad5). Higher frequencies of pre-immunization adenovirus-specific CD4⁺ T cells were associated with substantially decreased magnitude of HIV-specific CD4⁺ T cell responses and decreased breadth of HIV-specific CD8⁺ T cell responses in vaccine recipients, independent of type-specific preexisting Ad5-specific neutralizing antibody titers. Further, epitopes recognized by adenovirus-specific T cells were commonly conserved across many adenovirus serotypes, suggesting that cross-reactivity of preexisting adenovirus-specific T cells can extend to adenovirus vectors derived from rare serotypes. These findings provide what we believe to be a new understanding of how preexisting viral immunity may impact the efficacy of vaccines under current evaluation for prevention of HIV, tuberculosis, and malaria.

Safety and immunogenicity of an HIV adenoviral vector boost after DNA plasmid vaccine prime by route of administration: a randomized clinical trial.

BACKGROUND: In the development of HIV vaccines, improving immunogenicity while maintaining safety is critical. Route of administration can be an important factor. METHODOLOGY/PRINCIPAL FINDINGS: This multicenter, open-label, randomized trial, HVTN 069, compared routes of administration on safety and immunogenicity of a DNA vaccine prime given intramuscularly at 0, 1 and 2 months and a recombinant replication-defective adenovirus type 5 (rAd5) vaccine boost given at 6 months by intramuscular (IM), intradermal (ID), or subcutaneous (SC) route. Randomization was computer-generated by a central data management center; participants and staff were not blinded to group assignment. The outcomes were vaccine reactogenicity and humoral and cellular immunogenicity. Ninety healthy, HIV-1 uninfected adults in the US and Peru, aged 18-50 were enrolled and randomized. Due to the results of the Step Study, injections with rAd5 vaccine were halted; thus 61 received the booster dose of rAd5 vaccine (IM: 20; ID:21; SC:20). After the rAd5 boost, significant differences by study arm were found in severity of headache, pain and erythema/induration. Immune responses (binding and neutralizing antibodies, IFN-γ ELISpot HIV-specific responses and CD4+ and CD8+ T-cell responses by ICS) at four weeks after the rAd5 booster were not significantly different by administration route of the rAd5 vaccine boost (Binding antibody responses: IM: 66.7%; ID: 70.0%; SC: 77.8%; neutralizing antibody responses: IM: 11.1%; ID: 0.0%; SC 16.7%; ELISpot responses: IM: 46.7%; ID: 35.3%; SC: 44.4%; CD4+ T-cell responses: IM: 29.4%; ID: 20.0%; SC: 35.3%; CD8+ T-cell responses: IM: 29.4%; ID: 16.7%; SC: 50.0%.) CONCLUSIONS/SIGNIFICANCE: This study was limited by the reduced sample size. The higher frequency of local reactions after ID and SC administration and the lack of sufficient evidence to show that there were any differences in immunogenicity by route of administration do not support changing route of administration for the rAd5 boost. TRIAL REGISTRATION: ClinicalTrials.gov NCT00384787.

A phase I trial of preventive HIV vaccination with heterologous poxviral-vectors containing matching HIV-1 inserts in healthy HIV-uninfected subjects.

We evaluated replication-defective poxvirus vectors (modified vaccinia Ankara [MVA] and fowlpox [FPV]) in a homologous and heterologous vector prime-boost vaccination regimen containing matching HIV inserts (MVA-HIV and FPV-HIV) given at months 0, 1, 3, 5 and 7 in 150 healthy HIV-negative vaccinia-naïve participants. FPV-HIV alone was poorly immunogenic, while the high dose (10(9)pfu/2 ml) of MVA-HIV alone elicited maximal responses after two injections: CD4+ and CD8+ T-cell responses in 26/55 (47.3%) and 5/60 (8.3%) of participants, respectively, and IFN-γ ELISpot responses in 28/62 (45.2%). The infrequent CD8+ T-cell responses following MVA-HIV priming were boosted only by the heterologous (FPV-HIV) construct in 14/27 (51.9%) of participants post 4th vaccination. Alternatively, HIV envelope-specific binding antibodies were demonstrated in approximately two-thirds of recipients of the homologous boosting regimen, but in less than 20% of subjects after the heterologous vector boost. Thus, a heterologous poxvirus vector prime-boost regimen can induce HIV-specific CD8+ T-cell and CD4+ T-cell responses, which may be an important feature of an optimal regimen for preventive HIV vaccination.

Optimization of storage and shipment of cryopreserved peripheral blood mononuclear cells from HIV-infected and uninfected individuals for ELISPOT assays.

Functional immunologic assays using cryopreserved peripheral blood mononuclear cells (PBMC) are influenced by blood processing, storage and shipment. The objective of this study was to compare the viability, recovery and ELISPOT results of PBMC stored and shipped in liquid nitrogen (LN/LN) or stored in LN and shipped on dry ice (LN/DI) or stored at -70°C for 3 to 12 weeks and shipped on DI (70/DI 3 to 12); and to assess the effect of donor HIV infection status on the interaction between storage/shipment and the outcome measures. PBMC from 12 HIV-infected and 12 uninfected donors showed that LN/LN conferred higher viability and recovery than LN/DI or 70/DI 3, 6, 9 or 12. LN/DI PBMC had higher viability than any 70/DI PBMC. The PBMC viability and recovery linearly decreased with the duration of storage at -70°C from 3 to 12 weeks. This effect was more pronounced in samples from HIV-infected than uninfected donors. Results of ELISPOT assays using CMV pp65, CEF and Candida albicans antigens were qualitatively and quantitatively similar across LN/LN, LN/DI and 70/DI 3. However, ELISPOT values significantly decreased with the duration of storage at -70°C both in HIV-infected and uninfected donors. ELISPOT results also decreased with PBMC viability <70%.

A novel HIV T helper epitope-based vaccine elicits cytokine-secreting HIV-specific CD4+ T cells in a Phase I clinical trial in HIV-uninfected adults.

A Phase I human vaccine trial of a novel polypeptide vaccine of HIV T helper epitopes (EP-1043) and a DNA vaccine of HIV CTL epitopes was conducted in 84 healthy adult volunteers. The vaccine immunogenicity was assessed by an intracellular cytokine staining assay for IL-2, IL-4, TNF-alpha and IFN-gamma. Sixty eight percent (32/47) of subjects had a positive CD4+ T response after receiving two vaccinations of the polypeptide vaccine. The responding CD4+ T cells made various combinations of IL-2, IL-4, IFN-gamma, and TNF-alpha. The study demonstrated that the EP-1043 vaccine is safe, well-tolerated, and immunogenic.