Safety, tolerability, and immunogenicity of the chimpanzee adenovirus type 3-vectored Marburg virus (cAd3-Marburg) vaccine in healthy adults in the USA: a first-in-human, phase 1, open-label, dose-escalation trial.

BACKGROUND: WHO has identified Marburg virus as an emerging virus requiring urgent vaccine research and development, particularly due to its recent emergence in Ghana. We report results from a first-in-human clinical trial evaluating a replication-deficient recombinant chimpanzee adenovirus type 3 (cAd3)-vectored vaccine encoding a wild-type Marburg virus Angola glycoprotein (cAd3-Marburg) in healthy adults. METHODS: We did a first-in-human, phase 1, open-label, dose-escalation trial of the cAd3-Marburg vaccine at the Walter Reed Army Institute of Research Clinical Trials Center in the USA. Healthy adults aged 18-50 years were assigned to receive a single intramuscular dose of cAd3-Marburg vaccine at either 1 × 1010 or 1 × 1011 particle units (pu). Primary safety endpoints included reactogenicity assessed for the first 7 days and all adverse events assessed for 28 days after vaccination. Secondary immunogenicity endpoints were assessment of binding antibody responses and T-cell responses against the Marburg virus glycoprotein insert, and assessment of neutralising antibody responses against the cAd3 vector 4 weeks after vaccination. This study is registered with ClinicalTrials.gov, NCT03475056. FINDINGS: Between Oct 9, 2018, and Jan 31, 2019, 40 healthy adults were enrolled and assigned to receive a single intramuscular dose of cAd3-Marburg vaccine at either 1 × 1010 pu (n=20) or 1 × 1011 pu (n=20). The cAd3-Marburg vaccine was safe, well tolerated, and immunogenic. All enrolled participants received cAd3-Marburg vaccine, with 37 (93%) participants completing follow-up visits; two (5%) participants moved from the area and one (3%) was lost to follow-up. No serious adverse events related to vaccination occurred. Mild to moderate reactogenicity was observed after vaccination, with symptoms of injection site pain and tenderness (27 [68%] of 40 participants), malaise (18 [45%] of 40 participants), headache (17 [43%] of 40 participants), and myalgia (14 [35%] of 40 participants) most commonly reported. Glycoprotein-specific antibodies were induced in 38 (95%) of 40 participants 4 weeks after vaccination, with geometric mean titres of 421 [95% CI 209-846] in the 1 × 1010 pu group and 545 [276-1078] in the 1 × 1011 pu group, and remained significantly elevated at 48 weeks compared with baseline titres (39 [95% CI 13-119] in the 1 ×1010 pu group and 27 [95-156] in the 1 ×1011 pu group; both p<0·0001). T-cell responses to the glycoprotein insert and neutralising responses against the cAd3 vector were also increased at 4 weeks after vaccination. INTERPRETATION: This first-in-human trial of this cAd3-Marburg vaccine showed the agent is safe and immunogenic, with a safety profile similar to previously tested cAd3-vectored filovirus vaccines. 95% of participants produced a glycoprotein-specific antibody response at 4 weeks after a single vaccination, which remained in 70% of participants at 48 weeks. These findings represent a crucial step in the development of a vaccine for emergency deployment against a re-emerging pathogen that has recently expanded its reach to new regions. FUNDING: National Institutes of Health.

Multiplexed FluoroSpot for the Analysis of Dengue Virus- and Zika Virus-Specific and Cross-Reactive Memory B Cells.

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.

Effect of a Chikungunya Virus-Like Particle Vaccine on Safety and Tolerability Outcomes: A Randomized Clinical Trial.

Importance: Chikungunya virus (CHIKV) is a mosquito-borne Alphavirus prevalent 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 < .001) in the placebo group. Durability of the immune response was demonstrated through 72 weeks after vaccination. 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.

Safety, tolerability, and immunogenicity of two Zika virus DNA vaccine candidates in healthy adults: randomised, open-label, phase 1 clinical trials.

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.

Prior Dengue Virus Exposure Shapes T Cell Immunity to Zika Virus in Humans.

While progress has been made in characterizing humoral immunity to Zika virus (ZIKV) in humans, little is known regarding the corresponding T cell responses to ZIKV. Here, we investigate the kinetics and viral epitopes targeted by T cells responding to ZIKV and address the critical question of whether preexisting dengue virus (DENV) T cell immunity modulates these responses. We find that memory T cell responses elicited by prior infection with DENV or vaccination with tetravalent dengue attenuated vaccines (TDLAV) recognize ZIKV-derived peptides. This cross-reactivity is explained by the sequence similarity of the two viruses, as the ZIKV peptides recognized by DENV-elicited memory T cells are identical or highly conserved in DENV and ZIKV. DENV exposure prior to ZIKV infection also influences the timing and magnitude of the T cell response. ZIKV-reactive T cells in the acute phase of infection are detected earlier and in greater magnitude in DENV-immune patients. Conversely, the frequency of ZIKV-reactive T cells continues to rise in the convalescent phase in DENV-naive donors but declines in DENV-preexposed donors, compatible with more efficient control of ZIKV replication and/or clearance of ZIKV antigen. The quality of responses is also influenced by previous DENV exposure, and ZIKV-specific CD8 T cells from DENV-preexposed donors selectively upregulated granzyme B and PD1, unlike DENV-naive donors. Finally, we discovered that ZIKV structural proteins (E, prM, and C) are major targets of both the CD4 and CD8 T cell responses, whereas DENV T cell epitopes are found primarily in nonstructural proteins.IMPORTANCE The issue of potential ZIKV and DENV cross-reactivity and how preexisting DENV T cell immunity modulates Zika T cell responses is of great relevance, as the two viruses often cocirculate and Zika virus has been spreading in geographical regions where DENV is endemic or hyperendemic. Our data show that memory T cell responses elicited by prior infection with DENV recognize ZIKV-derived peptides and that DENV exposure prior to ZIKV infection influences the timing, magnitude, and quality of the T cell response. Additionally, we show that ZIKV-specific responses target different proteins than DENV-specific responses, pointing toward important implications for vaccine design against this global threat.

First-in-Human Evaluation of the Safety and Immunogenicity of an Intranasally Administered Replication-Competent Sendai Virus-Vectored HIV Type 1 Gag Vaccine: Induction of Potent T-Cell or Antibody Responses in Prime-Boost Regimens.

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: NCT01705990.

Assessment of the Safety and Immunogenicity of 2 Novel Vaccine Platforms for HIV-1 Prevention: A Randomized Trial.

BACKGROUND: A prophylactic HIV-1 vaccine is a global health priority. OBJECTIVE: To assess a novel vaccine platform as a prophylactic HIV-1 regimen. DESIGN: Randomized, double-blind, placebo-controlled trial. Both participants and study personnel were blinded to treatment allocation. (ClinicalTrials.gov: NCT01215149). SETTING: United States, East Africa, and South Africa. PATIENTS: Healthy adults without HIV infection. INTERVENTION: 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. MEASUREMENTS: Safety and immunogenicity and the effect of baseline vector immunity. RESULTS: 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. LIMITATIONS: 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. CONCLUSION: 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. PRIMARY FUNDING SOURCE: International AIDS Vaccine Initiative, National Institutes of Health, Ragon Institute, Crucell Holland.

A Phase 1 Study of 4 Live, Recombinant Human Cytomegalovirus Towne/Toledo Chimera Vaccines in Cytomegalovirus-Seronegative Men.

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.

Safety and Immunogenicity of a Randomized Phase 1 Prime-Boost Trial With ALVAC-HIV (vCP205) and Oligomeric Glycoprotein 160 From HIV-1 Strains MN and LAI-2 Adjuvanted in Alum or Polyphosphazene.

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.

Vaccine-elicited human T cells recognizing conserved protein regions inhibit HIV-1.

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 log10. 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.

Safety and immunogenicity of a trivalent virus-like particle vaccine against western, eastern, and Venezuelan equine encephalitis viruses: a phase 1, open-label, dose-escalation, randomised clinical trial.

BACKGROUND: 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. METHODS: 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. FINDINGS: 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 (PRNT80 geometric mean titre for EEEV 60·8, 95% CI 29·9-124·0; for VEEV 111·5, 49·8-249·8; and for WEEV 187·9, 90·0-392·2). Finally, 4 weeks after second vaccine administration, for all doses, the majority of trial participants developed an immune response to all three vaccine components (24 [83%] of 29 for EEEV; 26 [90%] of 29 for VEEV; 27 [93%] of 29 for WEEV; and 22 [76%] of 29 for EEEV, VEEV, and WEEV combined). INTERPRETATION: The 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. FUNDING: 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.

A phase 1/2 study of a multiclade HIV-1 DNA plasmid prime and recombinant adenovirus serotype 5 boost vaccine in HIV-Uninfected East Africans (RV 172).

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 .

Performance of International AIDS Vaccine Initiative African clinical research laboratories in standardised ELISpot and peripheral blood mononuclear cell processing in support of HIV vaccine clinical trials.

BACKGROUND: Standardisation of procedures for performing cellular functional assays across laboratories participating in multicentre clinical trials is key for generating comparable and reliable data. OBJECTIVE: This article describes the performance of accredited laboratories in Africa and Europe on testing done in support of clinical trials. METHODS: For enzyme-linked immunospot assay (ELISpot) proficiency, characterised peripheral blood mononuclear cells (PBMCs) obtained from 48 HIV-negative blood donors in Johannesburg, South Africa, were sent to participating laboratories between February 2010 and February 2014. The PBMCs were tested for responses against cytomegalovirus, Epstein Barr and influenza peptide pools in a total of 1751 assays. In a separate study, a total of 1297 PBMC samples isolated from healthy HIV-negative participants in clinical trials of two prophylactic HIV vaccine candidates in Kenya, Uganda, Rwanda and Zambia were analysed for cell viability, cell yield and cell recovery from frozen PBMCs. RESULTS: Most (99%) of the 1751 ELISpot proficiency assays had data within acceptable ranges with low responses to mock stimuli. No significant statistical difference were observed in ELISpot responses at the five laboratories actively conducting immunological analyses. Of the 1297 clinical trial PBMCs processed, 94% had cell viability above 90% and 96% had cell yield above 0.7 million per mL of blood in freshly isolated cells. All parameters were within the predefined acceptance criteria. CONCLUSION: We demonstrate that multiple laboratories can generate reliable, accurate and comparable data by using standardised procedures, having regular training, having regular equipment maintenance and using centrally sourced reagents.

Magnitude, breadth, and functional profile of T-cell responses during human immunodeficiency virus primary infection with B and BF viral variants.

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-gamma) 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.

Adeno-associated virus vectored immunoprophylaxis to prevent HIV in healthy adults: a phase 1 randomised controlled trial.

BACKGROUND: A preventive vaccine for HIV is a crucial public health need; adeno-associated virus (AAV)-mediated antibody gene delivery could be an alternative to immunisation to induce sustained expression of neutralising antibodies to prevent HIV. We assessed safety and tolerability of rAAV1-PG9DP, a recombinant AAV1 vector encoding the gene for PG9, a broadly neutralising antibody against HIV. METHODS: This first-in-human, proof-of-concept, double-blind, phase 1, randomised, placebo-controlled, dose-escalation trial was done at one clinical research centre in the UK. Healthy men aged 18-45 years without HIV infection were randomly assigned to receive intramuscular injection with rAAV1-PG9DP or placebo in the deltoid or quadriceps in one of four dose-escalating cohorts (group A, 4 × 1012 vector genomes; group B, 4 × 1013 vector genomes; group C, 8 × 1013 vector genomes; and group D, 1·2 × 1014 vector genomes). Volunteers were followed up for 48 weeks. The primary objective was to assess safety and tolerability. A secondary objective was to assess PG9 expression in serum and related HIV neutralisation activity. All volunteers were included in primary and safety analyses. The trial is complete and is registered with ClinicalTrials.gov, number NCT01937455. FINDINGS: Between Jan 30, 2014, and Feb 28, 2017, 111 volunteers were screened for eligibility. 21 volunteers were eligible and provided consent, and all 21 completed 48 weeks of follow-up. Reactogenicity was generally mild or moderate and resolved without intervention. No probably or definitely related adverse events or serious adverse events were recorded. We detected PG9 by HIV neutralisation in the serum of four volunteers, and by RT-PCR in muscle biopsy samples from four volunteers. We did not detect PG9 by ELISA in serum. PG9 anti-drug antibody was present in ten volunteers in the higher dose groups. Both anti-AAV1 antibodies and AAV1-specific T-cell responses were detected. INTERPRETATION: Future studies should explore higher doses of AAV, alternative AAV serotypes and gene expression cassettes, or other broadly neutralising HIV antibodies. FUNDING: International AIDS Vaccine Initiative, United States Agency for International Development, Bill & Melinda Gates Foundation, US National Institutes of Health.

A high viral burden predicts the loss of CD8 T-cell responses specific for subdominant gag epitopes during chronic human immunodeficiency virus infection.

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, r(2) = 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.

Varicella zoster virus--specific immune response after treatment with sodium stibogluconate for cutaneous leishmaniasis.

Sodium stibogluconate has been associated with the reactivation of varicella zoster virus (VZV) in otherwise healthy adults who receive the drug as treatment for cutaneous leishmaniasis. Ten patients receiving daily sodium stibogluconate underwent phlebotomy at baseline and at day 10. Flow cytometry-based immunophenotyping, VZV-specific IgG levels, and lymphocyte proliferative responses and intracellular cytokine secretion to VZV, cytomegalovirus, tetanus toxoid, superantigen, and mitogens were performed at both time points. The absolute number of total leukocytes, total lymphocytes, and lymphocyte subsets decreased overall without predilection for any particular subset of lymphocytes, such that the percentage of the total lymphocyte population for each lymphocyte subset did not change significantly (except for a marginal increase in percentage of cytotoxic T cells). Antibodies to VZV were measured in seven patients before and after treatment, and did not change. Lymphocyte proliferative responses to VZV and other antigens and mitogens did not change from baseline. The mechanism for the increased rate of VZV reactivation after treatment with sodium stibogluconate remains undefined.

In a mixed subtype epidemic, the HIV-1 Gag-specific T-cell response is biased towards the infecting subtype.

OBJECTIVES: Southwest Tanzania is affected by an HIV-1 epidemic consisting of subtypes A, C, and D, and their recombinant forms. This study was designed to assess whether the Gag- and Nef-specific T-cell response is biased towards recognizing the infecting subtype. METHODS: The infecting subtypes were characterized with a Multi-hybridization assay that discriminates between subtypes A, C and D. The interferon-gamma ELISPOT assay was used to detect the Gag- and Nef-specific T-cell responses in freshly isolated peripheral blood mononuclear cells in 56 seropositive patients. To study the HIV-specific T-cell responses, isolate-based Gag and Nef peptide sets representative of the locally occurring subtypes were used. The results were analysed at the total protein and single peptide level. RESULTS: In the study population, 35% were infected with a pure C subtype, 24% and 23% with ACD or AC recombinant forms, respectively. The total magnitude (P < 0.01) and breadth (P < 0.01) of the Gag-specific T-cell response detected with the subtype C-Gag peptide set was significantly greater than that detected with either the subtype A-Gag or D-Gag peptide sets. No significant difference was observed in the Nef-specific response. In 85% of responses targeting the most immunodominant Gag epitopes with subtype-specific sequence differences, the best recognized epitope variant corresponded to the infecting subtype. CONCLUSIONS: The Gag-specific T-cell response had a preference for recognizing peptides related to the infecting subtype.

Cryopreservation-related loss of antigen-specific IFNγ producing CD4+ T-cells can skew immunogenicity data in vaccine trials: Lessons from a malaria vaccine trial substudy.

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 3-5-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.

CTL epitope distribution patterns in the Gag and Nef proteins of HIV-1 from subtype A infected subjects in Kenya: use of multiple peptide sets increases the detectable breadth of the CTL response.

BACKGROUND: Subtype A is a major strain in the HIV-1 pandemic in eastern Europe, central Asia and in certain regions of east Africa, notably in rural Kenya. While considerable effort has been focused upon mapping and defining immunodominant CTL epitopes in HIV-1 subtype B and subtype C infections, few epitope mapping studies have focused upon subtype A. RESULTS: We have used the IFN-gamma ELIspot assay and overlapping peptide pools to show that the pattern of CTL recognition of the Gag and Nef proteins in subtype A infection is similar to that seen in subtypes B and C. The p17 and p24 proteins of Gag and the central conserved region of Nef were targeted by CTL from HIV-1-infected Kenyans. Several epitope/HLA associations commonly seen in subtype B and C infection were also observed in subtype A infections. Notably, an immunodominant HLA-C restricted epitope (Gag 296-304; YL9) was observed, with 8/9 HLA-CW0304 subjects responding to this epitope. Screening the cohort with peptide sets representing subtypes A, C and D (the three most prevalent HIV-1 subtypes in east Africa), revealed that peptide sets based upon an homologous subtype (either isolate or consensus) only marginally improved the capacity to detect CTL responses. While the different peptide sets detected a similar number of responses (particularly in the Gag protein), each set was capable of detecting unique responses not identified with the other peptide sets. CONCLUSION: Hence, screening with multiple peptide sets representing different sequences, and by extension different epitope variants, can increase the detectable breadth of the HIV-1-specific CTL response. Interpreting the true extent of cross-reactivity may be hampered by the use of 15-mer peptides at a single concentration and a lack of knowledge of the sequence that primed any given CTL response. Therefore, reagent choice and knowledge of the exact sequences that prime CTL responses will be important factors in experimentally defining cross-reactive CTL responses and their role in HIV-1 disease pathogenesis and validating vaccines aimed at generating broadly cross-reactive CTL responses.