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Western (WEEV), eastern (EEEV), and Venezuelan (VEEV) equine encephalitis viruses are mosquito-borne pathogens classified as potential biological warfare agents for which there are currently no approved human vaccines or therapies. We aimed to evaluate the safety, tolerability, and immunogenicity of an investigational trivalent virus-like particle (VLP) vaccine, western, eastern, and Venezuelan equine encephalitis (WEVEE) VLP, composed of WEEV, EEEV, and VEEV VLPs.The WEVEE VLP vaccine was evaluated in a phase 1, randomised, open-label, dose-escalation trial at the Hope Clinic of the Emory Vaccine Center at Emory University, Atlanta, GA, USA. Eligible participants were healthy adults aged 18-50 years with no previous vaccination history with an investigational alphavirus vaccine. Participants were assigned to a dose group of 6 μg, 30 μg, or 60 μg vaccine product and were randomly assigned (1:1) to receive the WEVEE VLP vaccine with or without aluminium hydroxide suspension (alum) adjuvant by intramuscular injection at study day 0 and at week 8. The primary outcomes were the safety and tolerability of the vaccine (assessed in all participants who received at least one administration of study product) and the secondary outcome was immune response measured as neutralising titres by plaque reduction neutralisation test (PRNT) 4 weeks after the second vaccination. This trial is registered at ClinicalTrials.gov, NCT03879603.Between April 2, 2019, and June 13, 2019, 30 trial participants were enrolled (mean age 32 years, range 21-48; 16 [53%] female participants and 14 [47%] male participants). Six groups of five participants each received 6 μg, 30 μg, or 60 μg vaccine doses with or without adjuvant, and all 30 participants completed study follow-up. Vaccinations were safe and well tolerated. The most frequently reported symptoms were mild injection-site pain and tenderness (22 [73%] of 30) and malaise (15 [50%] of 30). Dose-dependent differences in the frequency of pain and tenderness were found between the 6 μg, 30 μg, and 60 μg groups (p=0·0217). No significant differences were observed between dosing groups for any other reactogenicity symptom. Two adverse events (mild elevated blood pressure and moderate asymptomatic neutropenia) were assessed as possibly related to the study product in one trial participant (60 μg dose with alum); both resolved without clinical sequelae. 4 weeks after second vaccine administration, neutralising antibodies were induced in all study groups with the highest response seen against all three vaccine antigens in the 30 μg plus alum group (PRNTThe favourable safety profile and neutralising antibody responses, along with pressing public health need, support further evaluation of the WEVEE VLP vaccine in advanced-phase clinical trials.The Vaccine Research Center of the National Institute of Allergy and Infectious Diseases, National Institutes of Health funded the clinical trial. The US Department of Defense contributed funding for manufacturing of the study product.

Ex vivo functional immunoassays such as ELISpot and intracellular cytokine staining (ICS) by flow cytometry are crucial tools in vaccine development both in the identification of novel immunogenic targets and in the immunological assessment of samples from clinical trials. Cryopreservation and subsequent thawing of PBMCs via validated processes has become a mainstay of clinical trials due to processing restrictions inherent in the disparate location and capacity of trial centres, and also in the need to standardize biological assays at central testing facilities. Logistical and financial requirement to batch process samples from multiple study timepoints are also key. We used ELISpot and ICS assays to assess antigen-specific immunogenicity in blood samples taken from subjects enrolled in a phase II malaria heterologous prime-boost vaccine trial and showed that the freeze thaw process can result in a 3 to 5-fold reduction of malaria antigen-specific IFNγ-producing CD3+CD4+ effector populations from PBMC samples taken post vaccination. We have also demonstrated that peptide responsive CD8+ T cells are relatively unaffected, as well as CD4+ T cell populations that do not produce IFNγ. These findings contribute to a growing body of data that could be consolidated and synthesised as guidelines for clinical trials with the aim of increasing the efficiency of vaccine development pipelines.

Summary Background mAb114 is a single monoclonal antibody that targets the receptor-binding domain of Ebola virus glycoprotein, which prevents mortality in rhesus macaques treated after lethal challenge with Zaire ebolavirus . Here we present expedited data from VRC 608, a phase 1 study to evaluate mAb114 safety, tolerability, pharmacokinetics, and immunogenicity. Methods In this phase 1, dose-escalation study (VRC 608), conducted at the US National Institutes of Health (NIH) Clinical Center (Bethesda, MD, USA), healthy adults aged 18–60 years were sequentially enrolled into three mAb114 dose groups of 5 mg/kg, 25 mg/kg, and 50 mg/kg. The drug was given to participants intravenously over 30 min, and participants were followed for 24 weeks. Participants were only enrolled into increased dosing groups after interim safety assessments. Our primary endpoints were safety and tolerability, with pharmacokinetic and anti-drug antibody assessments as secondary endpoints. We assessed safety and tolerability in all participants who received study drug by monitoring clinical laboratory data and self-report and direct clinician assessment of prespecified infusion-site symptoms 3 days after infusion and systemic symptoms 7 days after infusion. Unsolicited adverse events were recorded for 28 days. Pharmacokinetic and anti-drug antibody assessments were completed in participants with at least 56 days of data. This trial is registered with ClinicalTrials.gov, number NCT03478891, and is active but no longer recruiting. Findings Between May 16, and Sept 27, 2018, 19 eligible individuals were enrolled. One (5%) participant was not infused because intravenous access was not adequate. Of 18 (95%) remaining participants, three (17%) were assigned to the 5 mg/kg group, five (28%) to the 25 mg/kg group, and ten (55%) to the 50 mg/kg group, each of whom received a single infusion of mAb114 at their assigned dose. All infusions were well tolerated and completed over 30–37 min with no infusion reactions or rate adjustments. All participants who received the study drug completed the safety assessment of local and systemic reactogenicity. No participants reported infusion-site symptoms. Systemic symptoms were all mild and present only in four (22%) of 18 participants across all dosing groups. No unsolicited adverse events occurred related to mAb114 and one serious adverse event occurred that was unrelated to mAb114. mAb114 has linear pharmacokinetics and a half-life of 24·2 days (standard error of measurement 0·2) with no evidence of anti-drug antibody development. Interpretation mAb114 was well tolerated, showed linear pharmacokinetics, and was easily and rapidly infused, making it an attractive and deployable option for treatment in outbreak settings. Funding Vaccine Research Center, US National Institute of Allergy and Infectious Diseases, and NIH.

Background. We report the first-in-human safety and immunogenicity assessment of a prototype intranasally administered, replication-competent Sendai virus (SeV)–vectored, human immunodeficiency virus type 1 (HIV-1) vaccine. Methods. Sixty-five HIV-1–uninfected adults in Kenya, Rwanda, and the United Kingdom were assigned to receive 1 of 4 prime-boost regimens (administered at 0 and 4 months, respectively; ratio of vaccine to placebo recipients, 12:4): priming with a lower-dose SeV-Gag given intranasally, followed by boosting with an adenovirus 35–vectored vaccine encoding HIV-1 Gag, reverse transcriptase, integrase, and Nef (Ad35-GRIN) given intramuscularly (SLA); priming with a higher-dose SeV-Gag given intranasally, followed by boosting with Ad35-GRIN given intramuscularly (SHA); priming with Ad35-GRIN given intramuscularly, followed by boosting with a higher-dose SeV-Gag given intranasally (ASH); and priming and boosting with a higher-dose SeV-Gag given intranasally (SHSH). Results. All vaccine regimens were well tolerated. Gag-specific IFN-γ enzyme-linked immunospot–determined response rates and geometric mean responses were higher (96% and 248 spot-forming units, respectively) in groups primed with SeV-Gag and boosted with Ad35-GRIN (SLA and SHA) than those after a single dose of Ad35-GRIN (56% and 54 spot-forming units, respectively) or SeV-Gag (55% and 59 spot-forming units, respectively); responses persisted for ≥8 months after completion of the prime-boost regimen. Functional CD8+ T-cell responses with greater breadth, magnitude, and frequency in a viral inhibition assay were also seen in the SLA and SHA groups after Ad35-GRIN boost, compared with those who received either vaccine alone. SeV-Gag did not boost T-cell counts in the ASH group. In contrast, the highest Gag-specific antibody titers were seen in the ASH group. Mucosal antibody responses were sporadic. Conclusions. SeV-Gag primed functional, durable HIV-specific T-cell responses and boosted antibody responses. The prime-boost sequence appears to determine which arm of the immune response is stimulated. Clinical Trials Registration. {"type":"clinical-trial","attrs":{"text":"NCT01705990","term_id":"NCT01705990"}}NCT01705990.

Background Human cytomegalovirus (HCMV) infection causes disease in newborns and transplant recipients. A HCMV vaccine (Towne) protects transplant recipients. Methods The genomes of Towne and the nonattenuated Toledo strain were recombined, yielding 4 Towne/Toledo chimera vaccines. Each of 36 HCMV-seronegative men received 1 subcutaneous dose of 10, 100, or 1000 plaque-forming units (PFU) in cohorts of 3. Safety and immunogenicity were evaluated over 12 weeks after immunization and for 52 weeks for those who seroconverted. Results There were no serious local or systemic reactions. No subject had HCMV in urine or saliva. For chimera 3, none of 9 subjects seroconverted. For chimera 1, 1 of 9 seroconverted (the seroconverter received 100 PFU). For chimera 2, 3 subjects seroconverted (1 received 100 PFU, and 2 received 1000 PFU). For chimera 4, 7 subjects seroconverted (1 received 10 PFU, 3 received 100 PFU, and 3 received 1000 PFU). All 11 seroconverters developed low but detectable levels of neutralizing activity. CD4+ T-cell responses were detectable in 1 subject (who received 100 PFU of chimera 4). Seven subjects receiving chimera 2 or 4 had detectable CD8+ T-cell responses to IE1; 3 responded to 1-2 additional antigens. Conclusions The Towne/Toledo chimera vaccine candidates were well tolerated and were not excreted. Additional human trials of chimeras 2 and 4 are appropriate. Clinical trials registration NCT01195571.

Background Prime-boost regimens comprising ALVAC-HIV (prime) and human immunodeficiency virus type 1 (HIV) Env (boost) induce HIV-specific neutralizing antibody and cell-mediated immune responses, but the impact of boost schedule and adjuvant requires further definition. Methods A phase 1 trial was conducted. In part A (open label), 19 volunteers received oligomeric glycoprotein 160 from HIV strains MN and LAI-2 (ogp160 MN/LAI-2) with dose escalation (25, 50, 100 μg) and either polyphosphazene (pP) or alum adjuvant. In part B, 72 volunteers received either placebo (n=12) or recombinant canarypox virus expressing HIV antigens (ALVAC-HIV [vCP205]) with different doses and schedules of ogp160 MN/LAI-2 in pP or alum (n = 60). Results The vaccines were safe and well tolerated, with no vaccine-related serious adverse events. Anti-gp70 V1V2 antibody responses were detected in 17 of 19 part A volunteers (89%) and 10%-100% of part B volunteers. Use of a peripheral blood mononuclear cell-based assay revealed that US-1 primary isolate neutralization was induced in 2 of 19 recipients of ogp160 protein alone (10.5%) and 5 of 49 prime-boost volunteers (10.2%). Among ogp160 recipients, those who received pP were more likely than those who received alum to have serum that neutralized tier 2 viruses (12% vs 0%; P = .015). Conclusions Administration of ogp160 with pP induces primary isolate tier 2 neutralizing antibody responses in a small percentage of volunteers, demonstrating proof of concept and underscoring the importance of further optimization of prime-boost strategies for HIV infection prevention. Clinical trials registration NCT00004579.

Importance Chikungunya virus (CHIKV) is a mosquito-borneAlphavirusprevalent worldwide. There are currently no licensed vaccines or therapies. Objective To evaluate the safety and tolerability of an investigational CHIKV virus–like particle (VLP) vaccine in endemic regions. Design, Setting, and Participants This was a randomized, placebo-controlled, double-blind, phase 2 clinical trial to assess the vaccine VRC-CHKVLP059-00-VP (CHIKV VLP). The trial was conducted at 6 outpatient clinical research sites located in Haiti, Dominican Republic, Martinique, Guadeloupe, and Puerto Rico. A total of 400 healthy adults aged 18 through 60 years were enrolled after meeting eligibility criteria. The first study enrollment occurred on November 18, 2015; the final study visit, March 6, 2018. Interventions Participants were randomized 1:1 to receive 2 intramuscular injections 28 days apart (20 µg, n = 201) or placebo (n = 199) and were followed up for 72 weeks. Main Outcomes and Measures The primary outcome was the safety (laboratory parameters, adverse events, and CHIKV infection) and tolerability (local and systemic reactogenicity) of the vaccine, and the secondary outcome was immune response by neutralization assay 4 weeks after second vaccination. Results Of the 400 randomized participants (mean age, 35 years; 199 [50%] women), 393 (98%) completed the primary safety analysis. All injections were well tolerated. Of the 16 serious adverse events unrelated to the study drugs, 4 (25%) occurred among 4 patients in the vaccine group and 12 (75%) occurred among 11 patients in the placebo group. Of the 16 mild to moderate unsolicited adverse events that were potentially related to the drug, 12 (75%) occurred among 8 patients in the vaccine group and 4 (25%) occurred among 3 patients in the placebo group. All potentially related adverse events resolved without clinical sequelae. At baseline, there was no significant difference between the effective concentration (EC50)—which is the dilution of sera that inhibits 50% infection in viral neutralization assay—geometric mean titers (GMTs) of neutralizing antibodies of the vaccine group (46; 95% CI, 34-63) and the placebo group (43; 95% CI, 32-57). Eight weeks following the first administration, the EC50 GMT in the vaccine group was 2005 (95% CI, 1680-2392) vs 43 (95% CI, 32-58;P Conclusions and Relevance Among healthy adults in a chikungunya endemic population, a virus-like particle vaccine compared with placebo demonstrated safety and tolerability. Phase 3 trials are needed to assess clinical efficacy. Trial Registration ClinicalTrials.gov Identifier:NCT02562482

Dengue virus (DENV) and Zika virus (ZIKV) are mosquito-borne pathogens that have a significant impact on human health. Immune sera, mAbs, and memory B cells (MBCs) isolated from patients infected with one DENV type can be cross-reactive with the other three DENV serotypes and even more distantly related flaviviruses such as ZIKV. Conventional ELISPOTs effectively measure Ab-secreting B cells but because they are limited to the assessment of a single Ag at a time, it is challenging to distinguish serotype-specific and cross-reactive MBCs in the same well. We developed a novel multifunction FluoroSpot assay using fluorescently labeled DENV and ZIKV (FLVs) that measures the cross-reactivity of Abs secreted by single B cells. Conjugation efficiency and recognition of FLVs by virus-specific Abs were confirmed by flow cytometry. Using a panel of DENV immune, ZIKV immune, and naive PBMC, FLVs were able to simultaneously detect DENV serotype-specific, ZIKV-specific, DENV serotype cross-reactive, and DENV/ZIKV cross-reactive Abs secreted by individual MBCs. Our findings indicate that the FLVs are sensitive and specific tools to detect specific and cross-reactive MBCs. These reagents will allow the assessment of the breadth as well as the durability of DENV/ZIKV B cell responses following vaccination or natural infection. This novel approach using FLVs in a FluoroSpot assay can be applied to other diseases such as influenza in which prior immunity with homosubtype- or heterosubtype-specific MBCs may influence subsequent infections.

Summary Background The Zika virus epidemic and associated congenital infections have prompted rapid vaccine development. We assessed two new DNA vaccines expressing premembrane and envelope Zika virus structural proteins. Methods We did two phase 1, randomised, open-label trials involving healthy adult volunteers. The VRC 319 trial, done in three centres, assessed plasmid VRC5288 (Zika virus and Japanese encephalitis virus chimera), and the VRC 320, done in one centre, assessed plasmid VRC5283 (wild-type Zika virus). Eligible participants were aged 18–35 years in VRC19 and 18–50 years in VRC 320. Participants were randomly assigned 1:1 by a computer-generated randomisation schedule prepared by the study statistician. All participants received intramuscular injection of 4 mg vaccine. In VRC 319 participants were assigned to receive vaccinations via needle and syringe at 0 and 8 weeks, 0 and 12 weeks, 0, 4, and 8 weeks, or 0, 4, and 20 weeks. In VRC 320 participants were assigned to receive vaccinations at 0, 4, and 8 weeks via single-dose needle and syringe injection in one deltoid or split-dose needle and syringe or needle-free injection with the Stratis device (Pharmajet, Golden, CO, USA) in each deltoid. Both trials followed up volunteers for 24 months for the primary endpoint of safety, assessed as local and systemic reactogenicity in the 7 days after each vaccination and all adverse events in the 28 days after each vaccination. The secondary endpoint in both trials was immunogenicity 4 weeks after last vaccination. These trials are registered with ClinicalTrials.gov, numbers NCT02840487 and NCT02996461. Findings VRC 319 enrolled 80 participants (20 in each group), and VRC 320 enrolled 45 participants (15 in each group). One participant in VRC 319 and two in VRC 320 withdrew after one dose of vaccine, but were included in the safety analyses. Both vaccines were safe and well tolerated. All local and systemic symptoms were mild to moderate. In both studies, pain and tenderness at the injection site was the most frequent local symptoms (37 [46%] of 80 participants in VRC 319 and 36 [80%] of 45 in VRC 320) and malaise and headache were the most frequent systemic symptoms (22 [27%] and 18 [22%], respectively, in VRC 319 and 17 [38%] and 15 [33%], respectively, in VRC 320). For VRC5283, 14 of 14 (100%) participants who received split-dose vaccinations by needle-free injection had detectable positive antibody responses, and the geometric mean titre of 304 was the highest across all groups in both trials. Interpretation VRC5283 was well tolerated and has advanced to phase 2 efficacy testing. Funding Intramural Research Program of the Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health.

The Interferon-gamma Enzyme-Linked ImmunoSpot (IFN-γ ELISpot) assay has been developed and used as an end-point assay in clinical trials for infectious diseases and cancer to detect the magnitude of antigen-specific immune responses. The ability to compare data generated by different laboratories across organizations is pivotal to understand the relative potency of different therapeutic and vaccine strategies. We developed an external proficiency program for the IFN-γ ELISpot assay that evaluates the laboratory performance based on five parameters: timeliness for data reporting; ability to handle cellular samples; detection of background (non-specific) responses; accuracy to consensus of the results; and precision of the measurements. Points are awarded for each criterion, and the sum of the points is used to determine a numeric and adjectival performance rating. Importantly, the evaluation of the accuracy to the consensus mean for the detection of antigen-specific responses using laboratory-specific procedures informs each laboratory and its sponsor on the degree of concordance of its results with those obtained by other laboratories. This study will ultimately provide the scientific community with information on how to organize and implement an external proficiency program to evaluate longitudinally the performance of the participating laboratories and, therefore, fulfill the requirements of the GCLP guidelines for laboratories performing end-point IFN-γ ELISpot assay for clinical trials.

Virus diversity and escape from immune responses are the biggest challenges to the development of an effective vaccine against HIV-1. We hypothesized that T-cell vaccines targeting the most conserved regions of the HIV-1 proteome, which are common to most variants and bear fitness costs when mutated, will generate effectors that efficiently recognize and kill virus-infected cells early enough after transmission to potentially impact on HIV-1 replication and will do so more efficiently than whole protein-based T-cell vaccines. Here, we describe the first-ever administration of conserved immunogen vaccines vectored using prime-boost regimens of DNA, simian adenovirus and modified vaccinia virus Ankara to uninfected UK volunteers. The vaccine induced high levels of effector T cells that recognized virus-infected autologous CD4 + cells and inhibited HIV-1 replication by up to 5.79 log 10. The virus inhibition was mediated by both Gag- and Pol- specific effector CD8 + T cells targeting epitopes that are typically subdominant in natural infection. These results provide proof of concept for using a vaccine to target T cells at conserved epitopes, showing that these T cells can control HIV-1 replication in vitro. © The American Society of Gene and Cell Therapy.

The gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay is used routinely to evaluate the potency of human immunodeficiency virus (HIV) vaccine candidates and other vaccine candidates. In order to compare candidates and pool data from multiple trial laboratories, validated standardized methods must be applied across laboratories. Proficiency panels are a key part of a comprehensive quality assurance program to monitor inter- and intralaboratory performance, as well as assay performance, over time. Seven International AIDS Vaccine Initiative-sponsored trial sites participated in the proficiency panels described in this study. At each laboratory, two operators independently processed identical sample sets consisting of frozen peripheral blood mononuclear cell (PBMC) samples from different donors by using four blind stimuli. PBMC recovery and viability after overnight resting and the IFN-γ ELISPOT assay performance were assessed. All sites demonstrated good performance in PBMC thawing and resting, with a median recovery of 78% and median viability of 95%. The laboratories were able to detect similar antigen-specific T-cell responses, ranging from 50 to >3,000 spot-forming cells per million PBMC. An approximate range of a half log in results from operators within or across sites was seen in comparisons of antigen-specific responses. Consistently low background responses were seen in all laboratories. The results of these proficiency panels demonstrate the ability of seven laboratories, located across three continents, to process PBMC samples and to rank volunteers with differential magnitudes of IFN-γ ELISPOT responses. These findings also illustrate the ability to standardize the IFN-γ ELISPOT assay across multiple laboratories when common training methods, reagents such as fetal calf serum, and standard operating procedures are adopted. These results are encouraging for laboratories that are using cell-based immunology assays to test HIV vaccines and other vaccines.

In this study, we assessed the feasibility of collecting standardized nasal and salivary samples at centers in Nairobi (Kenya), Kigali (Rwanda), and London (United Kingdom) using different collection devices and media (synthetic absorptive matrices versus flocked swabs, and Salimetrics oral swabs versus whole oral fluid collection). We detected anti-Gag (p24) and envelope (gp140) antibodies in both nasal fluid and salivary collections from all HIV-infected individuals, and cross-reactive anti-p24 antibodies were detected in 10% of HIV-uninfected individuals enrolled at one site. Collections from the nasal turbinates were comparable with samples collected deeper in the nasopharyngeal tract, and the yield of anti-p24 IgA in the whole oral fluid samples was higher than in samples collected from the parotid gland. We noted a trend toward reduced levels of anti-HIV antibody in the volunteers receiving anti-retroviral therapy. Levels of antibodies were stable over multiple collection visits. Overall, this study shows that nasal and salivary samples can be collected in a standardized manner over repeated visits in both low- and high-resource settings. These methods may be used in support for future HIV vaccine clinical trials.

Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.Objectives: The ability of HIV-1 vaccine candidates MRKAd5, VRC DNA/Ad5 and ALVAC/AIDSVAX to elicit CD8 + T cells with direct antiviral function was assessed and compared with HIV-1-infected volunteers. Design: Adenovirus serotype 5 (Ad5)-based regimens MRKAd5 and VRC DNA/Ad5, designed to elicit HIV-1-specific T cells, are immunogenic but failed to prevent infection or impact on viral loads in volunteers infected subsequently. Failure may be due in part to a lack of CD8 + T cells with effective antiviral functions. Methods: An in-vitro viral inhibition assay tested the ability of bispecific antibody expanded CD8 + T cells from peripheral blood mononuclear cells to inhibit replication of a multiclade panel of HIV-1 isolates in autologous CD4 + T cells. HIV-1 proteins recognized by CD8 + T cells were assessed by IFNγ enzyme-linked immunospot assay. Results: Ad5-based regimens elicited CD8 + T cells that inhibited replication of HIV-1 IIIB isolate with more limited inhibition of other isolates. IIIB isolate Gag and Pol genes have high sequence identities (>96%) to vector HIV-1 gene inserts, and these were the predominant HIV-1 proteins recognized by CD8 + T cells. Virus inhibition breadth was greater in antiretroviral naïve HIV-1-infected volunteers naturally controlling viremia (plasma viral load < 10 000/ml). HIV-1-inhibitory CD8 + T cells were not elicited by the ALVAC/AIDSVAX regimen. Conclusion: The Ad5-based regimens, although immunogenic, elicited CD8 + T cells with limited HIV-1-inhibition breadth. Effective T-cell-based vaccines should presumably elicit broader HIV-1-inhibition profiles. The viral inhibition assay can be used in vaccine design and to prioritize promising candidates with greater inhibition breadth for further clinical trials. ©

A prophylactic HIV-1 vaccine is a global health priority.To assess a novel vaccine platform as a prophylactic HIV-1 regimen.Randomized, double-blind, placebo-controlled trial. Both participants and study personnel were blinded to treatment allocation. (ClinicalTrials.gov: NCT01215149).United States, East Africa, and South Africa.Healthy adults without HIV infection.2 HIV-1 vaccines (adenovirus serotype 26 with an HIV-1 envelope A insert [Ad26.EnvA] and adenovirus serotype 35 with an HIV-1 envelope A insert [Ad35.Env], both administered at a dose of 5 × 1010 viral particles) in homologous and heterologous combinations.Safety and immunogenicity and the effect of baseline vector immunity.217 participants received at least 1 vaccination, and 210 (96%) completed follow-up. No vaccine-associated serious adverse events occurred. All regimens were generally well-tolerated. All regimens elicited humoral and cellular immune responses in nearly all participants. Preexisting Ad26- or Ad35-neutralizing antibody titers had no effect on vaccine safety and little effect on immunogenicity. In both homologous and heterologous regimens, the second vaccination significantly increased EnvA antibody titers (approximately 20-fold from the median enzyme-linked immunosorbent assay titers of 30-300 to 3000). The heterologous regimen of Ad26-Ad35 elicited significantly higher EnvA antibody titers than Ad35-Ad26. T-cell responses were modest and lower in East Africa than in South Africa and the United States.Because the 2 envelope inserts were not identical, the boosting responses were complex to interpret. Durability of the immune responses elicited beyond 1 year is unknown.Both vaccines elicited significant immune responses in all populations. Baseline vector immunity did not significantly affect responses. Second vaccinations in all regimens significantly boosted EnvA antibody titers, although vaccine order in the heterologous regimen had a modest effect on the immune response.International AIDS Vaccine Initiative, National Institutes of Health, Ragon Institute, Crucell Holland.

Background Human immunodeficiency virus (HIV) vaccine development remains a global priority. We describe the safety and immunogenicity of a multiclade DNA vaccine prime with a replication-defective recombinant adenovirus serotype 5 (rAd5) boost. Methods The vaccine is a 6-plasmid mixture encoding HIV envelope (env) subtypes A, B, and C and subtype B gag, pol, and nef, and an rAd5 expressing identical genes, with the exception of nef. Three hundred and twenty-four participants were randomized to receive placebo (n=138), a single dose of rAd5 at 10(10) (n = 24) or 10(11) particle units (n = 24), or DNA at 0, 1, and 2 months, followed by rAd5 at either 10(10) (n= 114) or 10(11) particle units (n = 24) boosting at 6 months. Participants were followed up for 24 weeks after the final vaccination. Results The vaccine was safe and well tolerated. HIV-specific T cell responses were detected in 63% of vaccinees. Titers of preexisting Ad5 neutralizing antibody did not affect the frequency and magnitude of T cell responses in prime-boost recipients but did affect the response rates in participants that received rAd5 alone (P = .037). Conclusion The DNA/rAd5 vaccination regimen was safe and induced HIV type 1 multi-clade T cell responses, which were not significantly affected by titers of preexisting rAd5 neutralizing antibody. Trial Registration. ClinicalTrials.gov identifier: NCT00123968 .

A recombinant modified vaccinia Ankara virus vaccine candidate (TBC-M4) expressing HIV-1 subtype C env, gag, tat-rev, and nef-RT genes was tested in a randomized, double-blind, dose escalation Phase I trial in 32 HIV-uninfected healthy volunteers who received three intramuscular injections of TBC-M4 at 0, 1, and 6 months of 5 x 10(7) plaque-forming units (pfu) (low dosage, LD) (n = 12) or 2.5 x 10(8) pfu (high dosage, HD) (n = 12) or placebo (n = 8). Local and systemic reactogenicity was experienced by approximately 67% and 83% of vaccine recipients, respectively. The reactogenicity events were mostly mild in severity. Severe but transient systemic reactogenicity was seen in one volunteer of the HD group. No vaccine-related serious adverse events or events suggesting perimyocarditis were seen. A higher frequency of local reactogenicity events was observed in the HD group. Cumulative HIV-specific IFN-gamma ELISPOT responses were detected in frozen PBMCs from 9/11 (82%), 12/12 (100%), and 1/8 (13%) volunteers after the third injection of the LD, HD, and placebo groups, respectively. Most of the responses were to gag and env proteins (maximum of 430 SFU/10(6) PBMCs) persisting across multiple time points. HIV-specific ELISA antibody responses were detected in 10/11, 12/12, and 0/8 volunteers post-third vaccination, in the LD, HD, and placebo groups, respectively. No neutralizing activity against HIV-1 subtype C isolates was detected. TBC-M4 appears to be generally safe and well-tolerated. The immune response detected was dose dependent, modest in magnitude, and directed mostly to env and gag proteins, suggesting further evaluation of this vaccine in a prime-boost regimen.

The presence of vector-specific immune responses may hamper the induction of responses to a foreign antigen encoded by the vector. We evaluated the impact of pre-existing immunity to vaccinia virus on the induction of HIV-specific responses after immunization of healthy volunteers with a HIV-1 DNA prime-MVA boost vaccine. Following three priming immunizations with HIV-1 DNA plasmids, the volunteers were boosted with a single injection of recombinant MVA encoding HIV-1 proteins. Pre-existing immunity to vaccinia virus did not reduce the proportion of individuals who responded to HIV-1, but did lower the magnitude of responses. Our results suggest that vaccinia-based vectors can be used to efficiently induce immune responses to vectored HIV-1 antigens, even in individuals with pre-existing immunity to vaccinia virus.

A human immunodeficiency virus (HIV) vaccine that limits disease and transmission is urgently needed. This clinical trial evaluated the safety and immunogenicity of an HIV vaccine that combines a plasmid-DNA priming vaccine and a modified vaccinia virus Ankara (MVA) boosting vaccine.Forty healthy volunteers were injected with DNA plasmids containing gp160 of HIV-1 subtypes A, B, and C; rev B; p17/p24 gag A and B, and RTmut B by use of a needle-free injection system. The vaccine was administered intradermally or intramuscularly, with or without recombinant granulocyte macrophage colony-stimulating factor, and boosted with a heterologous MVA containing env, gag, and pol of CRF01A_E. Immune responses were monitored with HIV-specific interferon (IFN)-gamma and interleukin (IL)-2 ELISpot and lymphoproliferative assays (LPAs).Vaccine-related adverse events were mild and tolerable. After receipt of the DNA priming vaccine, 11 (30%) of 37 vaccinees had HIV-specific IFN-gamma responses. After receipt of the MVA boosting vaccine, ELISpot assays showed that 34 (92%) of 37 vaccinees had HIV-specific IFN-gamma responses, 32 (86%) to Gag and 24 (65%) to Env. IFN-gamma production was detected in both the CD8(+) T cell compartment (5 of 9 selected vaccinees) and the CD4(+) T cell compartment (9 of 9). ELISpot results showed that 25 (68%) of 37 vaccinees had a positive IL-2 response and 35 (92%) of 38 had a positive LPA response. Of 38 subjects, a total of 37 (97%) were responders. One milligram of HIV-1 DNA administered intradermally was as effective as 4 mg administered intramuscularly in priming for the MVA boosting vaccine.This HIV-DNA priming-MVA boosting approach is safe and highly immunogenic.International Standard Randomised Controlled Trial number: ISRCTN32604572 .

The molecular pattern of the human immunodeficiency virus (HIV) epidemic in Argentina provides an appropriate scenario to study cellular immune responses in patients with non-clade B infection. We aimed to map T-cell responses in patients infected with BF recombinant variants and compare them with those of clade B patients. Sixteen recently infected patients were enrolled and grouped by viral subtype. Nef-specific responses were evaluated with a peptide matrix-based gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay using B and BF overlapping peptides. Cross-clade and clade-specific responses were found. A correlation between B versus BF Nef-specific responses was identified. Detailed analysis at the single-peptide level revealed that BF patients show a narrower response but greater magnitude. Nef immunodominant responses agreed with previous publications, although the B loop was targeted at an unexpectedly high frequency. The putative HLA allele(s) restricting each positive response was determined. Single-peptide level screening with two different peptide sets uncovered discordant responses (mostly caused by peptide offsetting) and allowed detection of increased breadth. Positive responses identified by ELISPOT assay were further studied by intracellular cytokine staining. These were almost exclusively mediated by CD8 T cells. Characterization of concordant responses revealed that cells show distinct functional profiles, depending on the peptide presented. Last, quality (in terms of polyfunctionality) of T cells was associated with better viral replication containment. Overall, interclade differences in the frequency of epitopes recognized, structural domains targeted, and magnitude of responses were identified. Screening T-cell responses with multiple sets increased sensitivity. Further support for the notion of polyfunctional CD8+T-cell requirement to better control viral replication is also provided.

Human immunodeficiency virus (HIV)-specific CD8 T-cell responses targeting products encoded within the Gag open reading frame have frequently been associated with better viral control and disease outcome during the chronic phase of HIV infection. To further clarify this relationship, we have studied the dynamics of Gag-specific CD8 T-cell responses in relation to plasma viral load and time since infection in 33 chronically infected subjects over a 9-month period. High baseline viral loads were associated with a net loss of breadth (P< 0.001) and a decrease in the total magnitude of the Gag-specific T-cell response in general (P= 0.03). Most importantly, the baseline viral load predicted the subsequent change in the breadth of Gag recognition over time (P< 0.0001,r2= 0.41). Compared to maintained responses, lost responses were low in magnitude (P< 0.0001) and subdominant in the hierarchy of Gag-specific responses. The present study indicates that chronic exposure of the human immune system to high levels of HIV viremia is a determinant of virus-specific CD8 T-cell loss.

The importance of HLA class I-restricted CD8 T-cell responses in the control of human immunodeficiency virus (HIV) infection is generally accepted. While several studies have shown an association of certain HLA class I alleles with slower disease progression, it is not fully established whether this effect is mediated by HIV-specific CD8 T-cell responses restricted by these alleles. In order to study the influence of the HLA class I alleles on the HIV-specific CD8 T-cell response and on viral control, we have assessed HIV-specific epitope recognition, plasma viral load, and expression of HLA class I alleles in a cohort of HIV-seropositive bar workers. Possession of the HLA class I alleles B5801, B8101, and B0702 was associated with a low median viral load and simultaneously with a broader median recognition of Gag epitopes compared to all other HLA alleles (twofold increase) (P= 0.0035). We further found an inverse linear relationship between the number of Gag epitopes recognized and the plasma viral load (R= −0.36;P= 0.0016). Particularly, recognition of multiple epitopes within two regions of Gag (amino acids [aa] 1 to 75 and aa 248 to 500) was associated with the maintenance of a low steady-state viremia, even years after acute infection.

HIV-1 strains containing subsubtype A2 are relatively rare in the pandemic but have been repeatedly identified in Kenya, where candidate vaccines based in part on subtype A, but not A2 strains, may be evaluated. Among the most recent is CRF16_A2D, a circulating recombinant form (CRF) whose prototypes are complete or partial HIV-1 sequences from Kenya, Korea, and Argentina. Using samples from blood bank discards in Kenya and complete genome sequencing, this report further documents CRF16_A2D and related recombinants and identifies a second CRF, CRF21_A2D. The two A2-containing CRFs, and two recombinants related to CRF16_A2D, share common structural elements but appear to have been independently derived. Concerted selection may have influenced the emergence and spread of certain A2-containing strains in Kenya. The second complete subtype C sequence from Kenya is also reported here. Monitoring of A2-containing recombinants and subtype C strains, both relatively rare in Kenya, may be informative in the course of cohort development and evaluation of candidate vaccines.

The HLA-B57 allele family is associated with slow progression to disease in HIV-1-infected individuals and restricts a potent CD8 response against the p24 protein. This study was designed to assess the sequence variation and the CD8 response against B57-restricted epitopes of the p24 protein in a cohort of HIV-1 subtype C-infected individuals possessing a high frequency of the B5703 allele. Gag sequences were amplified by PCR, cloned, and sequenced from 19 individuals including 8 B57-negative individuals. CD8 responses were assessed by interferon-gamma ELISPOT assay directly from PBMC using synthetic peptides matching the autologous virus as well as the peptides representing the sequence variants circulating within the B5703 individuals. The KF11 epitope (p24 amino acids 162-172) and variants of this epitope were immunodominant in subjects possessing the B5703 allele. Three variants were observed only in B5703 individuals. Differing patterns of cross-reactivity against variant peptides were observed and were dependent upon the sequence of the autologous virus. Subjects infected with the A2G, S4N variant of KF11 demonstrated poor cross-reactivity against all other variant peptides. Determination of the breadth of viral quasispecies circulating in a population provided crucial information for studying potential escape variants of an immunodominant epitope. The presented data show that the sequence of autologous virus is critical in determining the extent of cross-reactivity of a CD8 T cell response against heterologous virus variants. Efforts to optimize the cross-reactivity of vaccine-induced CD8 T cells may need to focus on the relative immunogenicity of minor sequence variation.

We have previously shown that adoptive transfer of in vitro CD3/CD28 activated autologous CD4(+) T cells results in increased CD4 counts and CD4/CD8 ratios in HIV+ subjects. In this report, analysis of variable beta (Vbeta) chain T cell receptor (TCR) repertoire showed that CD3/CD28 stimulation was able to increase polyclonality within skewed spectra types in vitro. In vivo, two of eight subjects showed increase in TCR diversity and importantly, in no subject did a highly skewed in vivo repertoire emerge. Measurement of proliferative response to alloantigen showed increases following infusions. Response to pharmacological stimulus and lectin via Interferon-gamma ELISpot assay showed increases in a subset of subjects following infusions. However, interferon-gamma response to HIV antigens and peptides declined concurrent with stable or diminishing latent infectious viral load in CD4(+) T cells. These data provide further evidence that adoptive transfer of activated autologous CD4(+) T cells can augment the immune system.

Identification of immune correlates associated with disease progression will provide information for HIV-1 vaccine design in countries such as Thailand, where the prevalent subtypes (B and CRF01_AE [E]) are characterized. In this study, plasma viral load and humoral immune responses were measured in 20 HIV-1 subtype E-infected Thai patients with different rates of disease progression, based on CD4(+) T cell decline and clinical symptoms. Nine progressors (PRs) and 11 slower progressors (SPs) were evaluated. CD4(+) T cell counts were inversely correlated with viral load (p = 0.004) and positively correlated with p24 Ab (p = 0.022). In progressors, p24 Ab showed a significant decrease (p0.001) over time. V3 and gp41 Ab did not change significantly in either group. Both CD4-binding site (CD4/gp120BS) and gp120 titers correlated positively with neutralizing antibody (NAb) against both a subtype E cell line-adapted virus (NP03) and a primary isolate (TH023). However, V3 Ab correlated only with NAb against NP03 (p0.001). Increased NAb over time was observed more frequently in SPs as compared with PRs, against both the TH023 (p = 0.004) and NPO3 (p = 0.004) viruses. Cross-clade antibody-dependent cellular cytotoxicity was demonstrated in both groups. These data suggest that in HIV-1 subtype E infection, declining p24 Ab titer is a predictive marker of disease progression, as described for subtype B. Furthermore, in subtype E-infected patients, slower progressors retain the immune competence to develop new antibody responses to Env over time; these evolving responses may contribute to prolonged survival during HIV-1 disease progression.

Administration of a clade C/B′ candidate HIV-1 DNA vaccine, ADVAX, by in vivo electroporation (EP) was safe and more immunogenic than intramuscular administration without EP. The breadth and specificity of T-cell responses to full-length Env were mapped. Responses to multiple Env regions were induced, with most focusing on V3/C4 and V2 regions, including the α4β7 integrin-binding domain. The breadth of responses induced by this DNA vaccine regimen was comparable to that of viral-vectored vaccine regimens.

Preclinical data are reported that support a human immunodeficiency virus (HIV) vaccine strategy using recombinant canarypox-HIV vectors (ALVAC-HIV) to load human dendritic cells (DCs) with HIV antigens. Clinical-grade DCs were infected with good manufacturing practice-grade ALVAC-HIV vaccine constructs. ALVAC infection, HIV gene expression, and DC viability and function were monitored by use of immunohistochemistry, flow cytometry, blastogenesis assays, antigen-specific interferon (IFN)-gamma enzyme-linked immunospot assay, and enzyme-linked immunosorbent assay protein detection. The vaccines infected both immature and mature DCs, and intracellular HIV-1 Gag protein was detected within hours. ALVAC-HIV induced DC maturation that was mediated by tumor necrosis factor-alpha and induced DC apoptosis that was directly related to the length of vaccine exposure. Of importance, the infected DCs remained functional in T cell stimulation assays and induced HIV antigen-specific CD8(+) T cell production of IFN-gamma from cells of HIV-1-infected individuals. These data support an ongoing HIV vaccine trial comparing conventional vaccine delivery routes with ex vivo vaccine-loaded autologous DCs for immunogenicity in HIV-1-uninfected volunteers.

Vaccines in general and HIV vaccines in particular are focusing ever more on the induction of cellular immunity specifically the generation of cytotoxic T cells (CTL). As progress is made towards developing a safe and effective HIV vaccine, there is a need for a robust, sensitive and reproducible assay to evaluate vaccine-induced cellular immunogenicity in Phase II/III trials. The enzyme-linked immunospot (ELISPOT) assay fits these criteria and is a technology that is readily transferable and amenable to high-through-put screening. There is a need for reagents that can be used as positive controls and for optimizing and standardizing the assay. We selected a panel of 23 8-11 mer Influenza virus (Flu), Cytomegalovirus (CMV) and Epstein Barr virus (EBV) epitopes recognized by CD8 positive T cells and presented by 11 class I HLA-A and HLA-B alleles whose cumulative frequencies represent >100% of Caucasian individuals. We examined interferon-gamma (IFN-gamma) secretion in peripheral blood mononuclear cells (PBMC) incubated with the peptides using a modified ELISPOT assay. IFN-gamma secretion was detected in 15/17 (88%) HIV-1 seronegative and 14/20 (70%) HIV-1 seropositive individuals. Release of IFN-gamma in response to the pool of peptides was CD8+ T cell mediated and HLA restricted. In vitro stimulation of PBMC with individual peptides or the pool of peptides led to the expansion of T cells capable of killing target cells expressing the appropriate viral antigen in a CTL assay. The size, shape and appearance of the spots produced using this peptide panel provided a standard for the establishment of acceptance criteria of spots for the evaluation of ELISPOT plates using an automated reader system.

Background. Defining the parameters that modulate vaccine responses in African populations will be imperative to design effective vaccines for protection against HIV, malaria, tuberculosis, and dengue virus infections. This study aimed to evaluate the contribution of the patient-specific immune microenvironment to the response to the licensed yellow fever vaccine 17D (YF-17D) in an African cohort. Methods. We compared responses to YF-17D in 50 volunteers in Entebbe, Uganda, and 50 volunteers in Lausanne, Switzerland. We measured the CD8+ T cell and B cell responses induced by YF-17D and correlated them with immune parameters analyzed by flow cytometry prior to vaccination. Results. We showed that YF-17D–induced CD8+ T cell and B cell responses were substantially lower in immunized individuals from Entebbe compared with immunized individuals from Lausanne. The impaired vaccine response in the Entebbe cohort associated with reduced YF-17D replication. Prior to vaccination, we observed higher frequencies of exhausted and activated NK cells, differentiated T and B cell subsets and proinflammatory monocytes, suggesting an activated immune microenvironment in the Entebbe volunteers. Interestingly, activation of CD8+ T cells and B cells as well as proinflammatory monocytes at baseline negatively correlated with YF-17D–neutralizing antibody titers after vaccination. Additionally, memory T and B cell responses in preimmunized volunteers exhibited reduced persistence in the Entebbe cohort but were boosted by a second vaccination. Conclusion. Together, these results demonstrate that an activated immune microenvironment prior to vaccination impedes efficacy of the YF-17D vaccine in an African cohort and suggest that vaccine regimens may need to be boosted in African populations to achieve efficient immunity. Trial registration. Registration is not required for observational studies. Funding. This study was funded by Canada’s Global Health Research Initiative, Defense Threat Reduction Agency, National Institute of Allergy and Infectious Diseases, Bill & Melinda Gates Foundation, and United States Agency for International Development.

A correlation between in vivo and in vitro virus control mediated by CD8+ T-cell populations has been demonstrated by CD8 T-cell-mediated inhibition of HIV-1 and SIV replication in vitro in peripheral blood mononuclear cells (PBMCs) from infected humans and non-human primates (NHPs), respectively. Here, the breadth and specificity of T-cell responses induced following vaccination with replication-defective adenovirus serotype 35 (Ad35) vectors containing a fusion protein of Gag, reverse transcriptase (RT), Integrase (Int) and Nef (Ad35-GRIN) and Env (Ad35-ENV), derived from HIV-1 subtype A isolates, was assessed in 25 individuals. The vaccine induced responses to a median of 4 epitopes per vaccinee. We correlated the CD8 responses to conserved vs. variable regions with the ability to inhibit a panel of 7 HIV-1 isolates representing multiple clades in a virus inhibition assay (VIA). The results indicate that targeting immunodominant responses to highly conserved regions of the HIV-1 proteome may result in an increased ability to inhibit multiple clades of HIV-1 in vitro. The data further validate the use of the VIA to screen and select future HIV vaccine candidates. Moreover, our data suggest that future T cell-focused vaccine design should aim to induce immunodominant responses to highly conserved regions of the virus.

Long-term safety is critical for the development and later use of a vaccine to prevent HIV/AIDS. Likewise, the persistence of vaccine-induced antibodies and their impact on HIV testing must be established. IAVI has sponsored several Phase I and IIA HIV vaccine trials enrolling healthy, HIV-seronegative African volunteers. Plasmid DNA and viral vector based vaccines were tested. No vaccine-related serious adverse events were reported. After completion of vaccine trials conducted between 2001–2007, both vaccine and placebo recipients were offered enrolment into an observational long-term follow-up study (LTFU) to monitor potential late health effects and persistence of immune responses. At scheduled 6-monthly clinic visits, a health questionnaire was administered; clinical events were recorded and graded for severity. Blood was drawn for HIV testing and cellular immune assays. 287 volunteers were enrolled; total follow-up after last vaccination was 1463 person years (median: 5.2 years). Ninety-three (93)% of volunteers reported good health at their last LTFU visit. Infectious diseases and injuries accounted for almost 50% of the 175 reported clinical events, of which over 95% were mild or moderate in severity. There were 30 six pregnancies, six incident HIV infections and 14 volunteers reported cases of social harm. Persistence of immune responses was rare. No safety signal was identified. No potentially vaccine-related medical condition, no immune mediated disease, or malignancy was reported. HIV vaccines studied in these trials had a low potential of induction of persisting HIV antibodies.

Emergence of SIV and HIV specific CD8 T cells has been shown to correlate with control of in vivo replication. Poor correlation between IFN-γ ELISPOT responses and in vivo control of the virus has triggered the development of more relevant assays to assess functional HIV-1 specific CD8 T-cell responses for the evaluation and prioritization of new HIV-1 vaccine candidates. We previously established a viral inhibition assay (VIA) that measures the ability of vaccine-induced CD8 T-cell responses to inhibit viral replication in autologous CD4 T cells. In this assay, viral replication is determined by measuring p24 in the culture supernatant. Here we describe the development of a novel VIA, referred to as IMC LucR VIA that exploits replication-competent HIV-1 infectious molecular clones (IMCs) in which the complete proviral genome is strain-specific and which express the Renilla luciferase (LucR) gene to determine viral growth and inhibition. The introduction of the luciferase readout does provide significant improvement of the read out time. In addition to switching to the LucR read out, changes made to the overall protocol resulted in the miniaturization of the assay from a 48 to a 96-well plate format, which preserved sample and allowed for the introduction of replicates. The overall assay time was reduced from 13 to 8. days. The assay has a high degree of specificity, and the previously observed non-specific background inhibition in cells from HIV-1 negative volunteers has been reduced dramatically. Importantly, we observed an increase in positive responses, indicating an improvement in sensitivity compared to the original VIA. Currently, only a limited number of "whole-genome" IMC-LucR viruses are available and our efforts will focus on expanding the panel to better evaluate anti-viral breadth. Overall, we believe the IMC LucR VIA provides a platform to assess functional CD8 T-cell responses in large-scale clinical trial testing, which will enhance the ability to select the most promising HIV-1 vaccine candidates capable of controlling HIV-1 replication in vivo. © 2013 The Authors.

Natural killer (NK) cells are being appreciated not only for their ability to recognize and lyse tumor cells and virus-infected cells but also for their immunoregulatory properties. NK cells provide a first line of defense against invading pathogens with a two pronged attack, lysis of infected cells and secretion of cytokines and chemokines with potent antipathogen effects. This article describes the standard chromium release assay, which measures the ability of NK cells derived from the peripheral blood to lyse appropriate target cells.

The human immunodeficiency virus type 1 (HIV-1) vpu gene encodes a type I anchored integral membrane phosphoprotein with two independent functions. First, it regulates virus release from a post-endoplasmic reticulum (ER) compartment by an ion channel activity mediated by its transmembrane anchor. Second, it induces the selective down regulation of host cell receptor proteins (CD4 and major histocompatibility complex class I molecules) in a process involving its phosphorylated cytoplasmic tail. In the present work, we show that the Vpu-induced proteolysis of nascent CD4 can be completely blocked by peptide aldehydes that act as competitive inhibitors of proteasome function and also by lactacystin, which blocks proteasome activity by covalently binding to the catalytic β subunits of proteasomes. The sensitivity of Vpu-induced CD4 degradation to proteasome inhibitors paralleled the inhibition of proteasome degradation of a model ubiquitinated substrate. Characterization of CD4-associated oligosaccharides indicated that CD4 rescued from Vpu-induced degradation by proteasome inhibitors is exported from the ER to the Golgi complex. This finding suggests that retranslocation of CD4 from the ER to the cytosol may be coupled to its proteasomal degradation. CD4 degradation mediated by Vpu does not require the ER chaperone calnexin and is dependent on an intact ubiquitin-conjugating system. This was demonstrated by inhibition of CD4 degradation (i) in cells expressing a thermally inactivated form of the ubiquitin-activating enzyme E 1 or (ii) following expression of a mutant form of ubiquitin (Lys 48 mutated to Arg 48 ) known to compromise ubiquitin targeting by interfering with the formation of polyubiquitin complexes. CD4 degradation was also prevented by altering the four Lys residues in its cytosolic domain to Arg, suggesting a role for ubiquitination of one or more of these residues in the process of degradation. The results clearly demonstrate a role for the cytosolic ubiquitin-proteasome pathway in the process of Vpu-induced CD4 degradation. In contrast to other viral proteins (human cytomegalovirus US2 and US11), however, whose translocation of host ER molecules into the cytosol occurs in the presence of proteasome inhibitors, Vpu-targeted CD4 remains in the ER in a transport-competent form when proteasome activity is blocked.

Activation of peripheral blood CD4 + helper T lymphocytes establishes a permissive state for growth of HIV-1. Activated T lymphocytes expressed increased sialidase (neuraminidase) activity and were hyposialylated. Treatment of freshly isolated peripheral blood mononuclear cells (PBMCs) with microbial neuraminidase (NANase) or phytohemagglutinin (PHA) prior to infection at low multiplicity with T cell line-adapted HIV-1 IIIB resulted in production of large amounts of p24 antigen and reverse transcriptase. In contrast, neither viral component was detected in the medium of mock-treated cells infected at a similar multiplicity through 21 days in culture. The titer of a stock solution of HIV-1 IIIB was 1.4 ± 0.18 log 10 greater in NANase-treated PBMCs than in mock-treated cells; the titer was similarly raised 1.5 to 1.76 ± 0.18 log 10 in PHA-treated cells. Growth of the primary isolate HIV-1 91/US/056 was also enhanced in NANase-treated PBMCs; the titer of a stock solution of HIV-1 91/US/056 was 1.0 ± 0.16 log 10 greater in NANase-treated PBMCs than in mock-treated cells 7 days after infection. No enhancement of viral growth in PBMCs was detected when NANase was heat-inactivated or specifically inhibited with 2,3-dehydro-2-desoxy- N -acetyl-neuraminic acid prior to use. Treatment of PBMCs with NANase did not alter the distribution of lymphocyte subsets nor change the density of CD4 antigen per cell after 7 days in culture. Whereas PHA treatment of PBMCs was mitogenic, pretreatment with NANase was not; the amount of [ 3 H]thymidine incorporated into DNA and culture growth characteristics were similar for NANase- and mock-treated cells. Thus, desialylation of PBMCs promoted a permissive state for growth of HIV-1 without affecting the rate of DNA synthesis or relative number of target CD4 + cells.

A randomized, double-blind, placebo-controlled phase I trial was conducted in 32 HIV-uninfected healthy volunteers to assess the safety and immunogenicity of 3 doses of DNA vaccine (Advax) plus 1 dose of recombinant modified vaccinia virus Ankara (MVA) (TBC-M4) or 3 doses of TBC-M4 alone (groups A and B, respectively). Both vaccine regimens were found to be safe and well tolerated. Gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISPOT) assay responses were detected in 1/10 (10%) individuals in group A after three Advax primes and in 9/9 individuals (100%) after the MVA boost. In group B, IFN-γ ELISPOT responses were detected in 6/12 (50%) and 7/11 (64%) individuals after the second and third MVA vaccinations, respectively. Responses to all vaccine components, but predominantly to Env, were seen. The breadth and magnitude of the T cell response and viral inhibition were greater in group A than in group B, indicating that the quality of the T-cell response was enhanced by the DNA prime. Intracellular cytokine staining indicated that the T-cell responses were polyfunctional but were skewed toward Env with a CD4+phenotype. At 2 weeks after the last vaccination, HIV-specific antibody responses were detected in all (100%) group B and 1/11 (9.1%) group A vaccinees. Vaccinia virus-specific responses were detected in all (100%) group B and 2/11 (18.2%) group A vaccinees. In conclusion, HIV-specific T-cell responses were seen in the majority of volunteers in groups A and B but with a trend toward greater quality of the T-cell response in group A. Antibody responses were better in group B than in group A.

We characterized prime-boost vaccine regimens using heterologous and homologous vector and gene inserts. Heterologous regimens offer a promising approach that focuses the cell-mediated immune response on the insert and away from vector-dominated responses. Ad35-GRIN/ENV (Ad35-GE) vaccine is comprised of two vectors containing sequences from HIV-1 subtype A gag, rt, int, nef (Ad35-GRIN) and env (Ad35-ENV). MVA-CMDR (MVA-C), MVA-KEA (MVA-K) and MVA-TZC (MVA-T) vaccines contain gag, env and pol genes from HIV-1 subtypes CRF01_AE, A and C, respectively. Balb/c mice were immunized with different heterologous and homologous vector and insert prime-boost combinations. HIV and vector-specific immune responses were quantified post-boost vaccination. Gag-specific IFN-γ ELISPOT, intracellular cytokine staining (ICS) (CD107a, IFN-γ, TNF-α and IL-2), pentamer staining and T-cell phenotyping were used to differentiate responses to inserts and vectors. Ad35-GE prime followed by boost with any of the recombinant MVA constructs (rMVA) induced CD8+ Gag-specific responses superior to Ad35-GE-Ad35-GE or rMVA-rMVA prime-boost combinations. Notably, there was a shift toward insert-focus responses using heterologous vector prime-boost regimens. Gag-specific central and effector memory T cells were generated more rapidly and in greater numbers in the heterologous compared to the homologous prime-boost regimens. These results suggest that heterologous prime-boost vaccination regimens enhance immunity by increasing the magnitude, onset and multifunctionality of the insert-specific cell-mediated immune response compared to homologous vaccination regimens. This study supports the rationale for testing heterologous prime-boost regimens in humans.

The genetic diversity of HIV-1 across the globe is a major challenge for developing an HIV vaccine. To facilitate immunogen design, it is important to characterize clusters of commonly targeted T-cell epitopes across different HIV clades. To address this, we examined 39 HIV-1 clade C infected individuals for IFN-γ Gag-specific T-cell responses using five sets of overlapping peptides, two sets matching clade C vaccine candidates derived from strains from South Africa and China, and three peptide sets corresponding to consensus clades A, B, and D sequences. The magnitude and breadth of T-cell responses against the two clade C peptide sets did not differ, however clade C peptides were preferentially recognized compared to the other peptide sets. A total of 84 peptides were recognized, of which 19 were exclusively from clade C, 8 exclusively from clade B, one peptide each from A and D and 17 were commonly recognized by clade A, B, C and D. The entropy of the exclusively recognized peptides was significantly higher than that of commonly recognized peptides (p = 0.0128) and the median peptide processing scores were significantly higher for the peptide variants recognized versus those not recognized (p = 0.0001). Consistent with these results, the predicted Major Histocompatibility Complex Class I IC(50) values were significantly lower for the recognized peptide variants compared to those not recognized in the ELISPOT assay (p

Background: DNA-based vaccines have been safe but weakly immunogenic in humans to date. Methods and Findings: We sought to determine the safety, tolerability, and immunogenicity of ADVAX, a multigenic HIV-1 DNA vaccine candidate, injected intramuscularly by in vivo electroporation (EP) in a Phase-1, double-blind, randomized placebo-controlled trial in healthy volunteers. Eight volunteers each received 0.2 mg, 1 mg, or 4 mg ADVAX or saline placebo via EP, or 4 mg ADVAX via standard intramuscular injection at weeks 0 and 8. A third vaccination was administered to eleven volunteers at week 36. EP was safe, well-tolerated and considered acceptable for a prophylactic vaccine. EP delivery of ADVAX increased the magnitude of HIV-1-specific cell mediated immunity by up to 70-fold over IM injection, as measured by gamma interferon ELISpot. The number of antigens to which the response was detected improved with EP and increasing dosage. Intracellular cytokine staining analysis of ELISpot responders revealed both CD4+ and CD8+ T cell responses, with co-secretion of multiple cytokines. Conclusions: This is the first demonstration in healthy volunteers that EP is safe, tolerable, and effective in improving the magnitude, breadth and durability of cellular immune responses to a DNA vaccine candidate. Trial Registration: ClinicalTrials.gov NCT00545987

Background: The Comprehensive T Cell Vaccine Immune Monitoring Consortium (CTC-VIMC) was created to provide standardized immunogenicity monitoring services for HIV vaccine trials. The ex vivo interferon-gamma (IFN-c) ELISpot is used extensively as a primary immunogenicity assay to assess T cell-based vaccine candidates in trials for infectious diseases and cancer. Two independent, GCLP-accredited central laboratories of CTC-VIMC routinely use their own standard operating procedures (SOPs) for ELISpot within two major networks of HIV vaccine trials. Studies are imperatively needed to assess the comparability of ELISpot measurements across laboratories to benefit optimal advancement of vaccine candidates. Methods: We describe an equivalence study of the two independently qualified IFN-g ELISpot SOPs. The study design, data collection and subsequent analysis were managed by independent statisticians to avoid subjectivity. The equivalence of both response rates and positivity calls to a given stimulus was assessed based on pre-specified acceptance criteria derived from a separate pilot study. Findings: Detection of positive responses was found to be equivalent between both laboratories. The 95% C.I. on the difference in response rates, for CMV (21.5%, 1.5%) and CEF (20.4%, 7.8%) responses, were both contained in the prespecified equivalence margin of interval [215%, 15%]. The lower bound of the 95% C.I. on the proportion of concordant positivity calls for CMV (97.2%) and CEF (89.5%) were both greater than the pre-specified margin of 70%. A third CTC-VIMC central laboratory already using one of the two SOPs also showed comparability when tested in a smaller sub-study. Interpretation: The described study procedure provides a prototypical example for the comparison of bioanalytical methods in HIV vaccine and other disease fields. This study also provides valuable and unprecedented information for future vaccine candidate evaluations on the comparison and pooling of ELISpot results generated by the CTC-VIMC central core laboratories.

The recombinant vaccine, tgAAC09, based on an adeno-associated virus serotype 2 (AAV2) vector encoding HIV-1 subtype C Gag, protease, and part of reverse transcriptase, induced robust T cell and antibody responses in nonhuman primates. In a previous phase I study in 80 healthy HIV-seronegative European and Indian adults, the vaccine was generally safe, well tolerated, and modestly immunogenic when administered once at doses up to 3 x 10(11) DRP. This phase II double-blind, randomized, placebo-controlled trial tested two administrations and a higher dosage of tgAAC009. Ninety-one healthy HIV-seronegative adults from three African countries were given one of three dosage levels of tgAAC09 (3 x 10(10), 3 x 10(11), or 3 x 10(12) DRP) intramuscularly, either at a 6- or 12-month interval; follow-up was 18 months. Overall, 65% and 57% of vaccine recipients experienced local and systemic signs and symptoms, respectively, most being mild. Frequency and severity were not dose related and were similar to those in placebo recipients. No vaccine-related serious adverse events were reported. Overall, HIV-specific T cell responses were detected by IFN-gamma ELISPOT in 17/69 (25%) vaccine recipients with 38% (10/26) responders in the highest dosage group. The response rate improved significantly with boosting at 6, but not 12 months, in the 3 x 10(11) and 3 x 10(12) dosage groups only. Neutralizing antibody titers to the AAV2 did not alter the frequency of immune responses to HIV. Two doses of tgAAC09 were well tolerated at the dosage levels given. Fewer than half the recipients of the highest vaccine dosage, 3 x 10(12) DRP, had T cell responses to HIV.

Human immunodeficiency virus type 1 (HIV-1) vaccine and natural history studies are critically dependent on the ability to isolate, cryopreserve, and thaw peripheral blood mononuclear cell (PBMC) samples with a high level of quality and reproducibility. Here we characterize the yield, viability, phenotype, and function of PBMC from HIV-1-infected and uninfected Ugandans and describe measures to ascertain reproducibility and sample quality at the sites that perform cryopreservation. We have developed a comprehensive internal quality control program to monitor processing, including components of method validation. Quality indicators for real-time performance assessment included the time from venipuncture to cryopreservation, time for PBMC processing, yield of PBMC from whole blood, and viability of the PBMC before cryopreservation. Immune phenotype analysis indicated lowered B-cell frequencies following processing and cryopreservation for both HIV-1-infected and uninfected subjects (P< 0.007), but all other major lymphocyte subsets were unchanged. Long-term cryopreservation did not impact function, as unstimulated specimens exhibited low background and all specimens responded to staphylococcal enterotoxin B (SEB) by gamma interferon and interleukin-2 production, as measured by intracellular cytokine staining. Samples stored for more than 3 years did not decay with regard to yield or viability, regardless of HIV-1 infection status. These results demonstrate that it is possible to achieve the high level of quality necessary for vaccine trials and natural history studies in a resource-limited setting and provide strategies for laboratories to monitor PBMC processing performance.

Background We conducted a Phase I dose-escalation trial of ADMVA, a Clade-B'/C-based HIV-1 candidate vaccine expressing env, gag, pol, nef, and tat in a modified vaccinia Ankara viral vector. Sequences were derived from a prevalent circulating HIV-1 recombinant form in Yunnan, China, an area of high HIV incidence. The objective was to evaluate the safety and immunogenicity of ADMVA in human volunteers. Methodology/Principal Findings ADMVA or placebo was administered intramuscularly at months 0, 1 and 6 to 50 healthy adult volunteers not at high risk for HIV-1. In each dosage group [1×107 (low), 5×107 (mid), or 2.5×108 pfu (high)] volunteers were randomized in a 3∶1 ratio to receive ADMVA or placebo in a double-blinded design. Subjects were followed for local and systemic reactogenicity, adverse events including cardiac adverse events, and clinical laboratory parameters. Study follow up was 18 months. Humoral immunogenicity was evaluated by anti-gp120 binding ELISA, immunoflourescent staining, and HIV-1 neutralization. Cellular immunogenicity was assessed by a validated IFNγ ELISpot assay and intracellular cytokine staining. Anti-vaccinia binding titers were measured by ELISA. ADMVA was generally well-tolerated, with no vaccine-related serious adverse events or cardiac adverse events. Local or systemic reactogenicity events were reported by 77% and 78% of volunteers, respectively. The majority of events were of mild intensity. The IFNγ ELISpot response rate to any HIV antigen was 0/12 (0%) in the placebo group, 3/12 (25%) in the low dosage group, 6/12 (50%) in the mid dosage group, and 8/13 (62%) in the high dosage group. Responses were often multigenic and occasionally persisted up to one year post vaccination. Antibodies to gp120 were detected in 0/12 (0%), 8/13 (62%), 6/12 (50%) and 10/13 (77%) in the placebo, low, mid, and high dosage groups, respectively. Antibodies persisted up to 12 months after vaccination, with a trend toward agreement with the ability to neutralize HIV-1 SF162 in vitro. Two volunteers mounted antibodies that were able to neutralize clade-matched viruses. Conclusions/Significance ADMVA was well-tolerated and elicited durable humoral and cellular immune responses. Trial Registration Clinicaltrials.gov NCT00252148

Background We conducted a Phase I dose escalation trial of ADVAX, a DNA-based candidate HIV-1 vaccine expressing Clade C/B' env, gag, pol, nef, and tat genes. Sequences were derived from a prevalent circulating recombinant form in Yunnan, China, an area of high HIV-1 incidence. The objective was to evaluate the safety and immunogenicity of ADVAX in human volunteers. Methodology/Principal Findings ADVAX or placebo was administered intramuscularly at months 0, 1 and 3 to 45 healthy volunteers not at high risk for HIV-1. Three dosage levels [0.2 mg (low), 1.0 mg (mid), and 4.0 mg (high)] were tested. Twelve volunteers in each dosage group were assigned to receive ADVAX and three to receive placebo in a double-blind design. Subjects were followed for local and systemic reactogenicity, adverse events, and clinical laboratory parameters. Study follow up was 18 months. Humoral immunogenicity was evaluated by anti-gp120 binding ELISA. Cellular immunogenicity was assessed by a validated IFNγ ELISpot assay and intracellular cytokine staining. ADVAX was safe and well-tolerated, with no vaccine-related serious adverse events. Local and systemic reactogenicity events were reported by 64% and 42% of vaccine recipients, respectively. The majority of events were mild. The IFNγ ELISpot response rates to any HIV antigen were 0/9 (0%) in the placebo group, 3/12 (25%) in the low-dosage group, 4/12 (33%) in the mid-dosage group, and 2/12 (17%) in the high-dosage group. Overall, responses were generally transient and occurred to each gene product, although volunteers responded to single antigens only. Binding antibodies to gp120 were not detected in any volunteers, and HIV seroconversion did not occur. Conclusions/Significance ADVAX delivered intramuscularly is safe, well-tolerated, and elicits modest but transient cellular immune responses. Trial Registration Clinicaltrials.gov NCT00249106

Recombinant modified vaccinia virus Ankara (rMVA) expressing HIV-1 genes are promising vaccine candidates. Toward the goal of conducting clinical trials with one or a cocktail of recombinant viruses, four rMVAs expressing env and gag-pol genes from primary HIV-1 isolates representing predominant subtypes from Kenya, Tanzania, Uganda, and Thailand (A, C, D, and CRF01_AE, respectively) were constructed. Efficient expression, processing, and function of Env and Gag were demonstrated. All inserted genes were shown to be genetically stable after repeated passage in cell culture. Strong HIV-specific cellular and humoral immune responses were elicited in mice immunized with each individual vaccine candidate. The MVA/CMDR vaccine candidate expressing CRF01_AE genes has elicited HIV-specific T-cell responses in two independent Phase I clinical trials. Further testing of the other rMVA is warranted.

Marcella Sarzotti-Kelsoe and colleagues harmonize various approaches to Good Clinical Laboratory Practice for clinical trials into a single set of recommendations.

The advent of T-cell assay methodologies that are amenable to high throughput coupled with the availability of large libraries of overlapping peptides have revolutionized the fields of vaccine efficacy testing and cellular immune response assessment. Since T-cell assay performance is critically dependent upon the quality and specificity of the stimulating peptides, assurance of high-quality and reliable input peptides is an important aspect of assay validation. Herein, we demonstrate that individual peptides from large human immunodeficiency virus (HIV)-based peptide library sets obtained directly from two independent custom peptide suppliers contained contaminating peptides capable of giving false-positive results, which were consistent with nominal antigen-specific CD8 + T-cell responses. In-depth investigation of the cellular response in terms of responding CD8 + T-cell frequency and human leukocyte antigen (HLA) restriction led to the conclusion that one set of HIV type 1 (HIV-1)-derived peptides was contaminated with a peptide from human cytomegalovirus (HCMV), which is commonly used in cellular immunology research applications. Analytical characterization of the original stock of the suspect HIV-1 peptide confirmed the presence of ∼1% by weight of the HCMV peptide. These observations have critical implications for quality assurance (QA) and quality control (QC) of peptides used in clinical trials where cellular immune-based assays are important end-point determinants. We propose a simple schema of biological QA/QC protocols to augment the standard biochemical QA/QC analyses as a means to circumvent this and other problems that can affect cellular immune-based assay outcome and interpretation.

The results presented here are from the preclinical evaluation in BALB/c mice of a DNA prime/modified vaccinia virus Ankara (MVA) boost multi-gene multi-subtype human immunodeficiency virus-1 (HIV-1) vaccine intended for use in humans. The plasmid DNA vaccine was delivered intradermally using a Biojector, and the MVA was delivered intramuscularly by needle. This combination of recombinant DNA and MVA proved to induce extraordinarily strong cellular responses, with more than 80% of the CD8(+) T cells specific for HIV-1 antigens. Furthermore, we show that the DNA priming increases the number of T-cell epitopes recognized after the MVA boost. In the prime/boost-immunized animals, a significant proportion of CD8(+) T cells were stained positive for both interferon-gamma (IFN-gamma) and interleukin-2 (IL-2), a feature that has been associated with control of HIV-1 infection in long-term non-progressors. The HIV-1-specific antibody levels were moderate after the plasmid DNA immunizations but increased dramatically after the MVA boost. Although the initial injection of MVA induced significant levels of vaccinia-neutralizing antibodies, the HIV-specific responses were still significantly boosted by the second MVA immunization. The results from this study demonstrate the potency of this combination of DNA plasmids and MVA construct to induce broad and high levels of immune responses against several HIV-1 proteins of different subtypes.