Viral inhibition assay: a CD8 T cell neutralization assay for use in clinical trials of HIV-1 vaccine candidates.

We have characterized an assay measuring CD8 T cell-mediated inhibition of human immunodeficiency virus (HIV) type 1 replication, demonstrating specificity and reproducibility and employing a panel of primary HIV-1 isolates. The assay uses relatively simple autologous cell culture and enzyme-linked immunosorbent assay, avoids generation of T cell clones, and can be performed with <2 million peripheral blood mononuclear cells. Efficient CD8 T cell-mediated cross-clade inhibition of HIV-1 replication in vitro was demonstrated in antiretroviral therapy-naive HIV-1-infected subjects with controlled viral replication in vivo but not in viremic subjects. An HIV-1 vaccine candidate, consisting of DNA and recombinant adenovirus 5 vectors tested in a phase I clinical trial, induced CD8 T cells that efficiently inhibited HIV-1 in a HLA-I-dependent manner. Assessment of direct antiviral T cell function by this assay provides additional information to guide vaccine design and the prioritizing of candidates for further clinical trials.

Broad HIV epitope specificity and viral inhibition induced by multigenic HIV-1 adenovirus subtype 35 vector vaccine in healthy uninfected adults.

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.

Safety and immunogenicity of DNA prime and modified vaccinia ankara virus-HIV subtype C vaccine boost in healthy adults.

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.

A DNA-based candidate HIV vaccine delivered via in vivo electroporation induces CD4 responses toward the α4ß7-binding V2 loop of HIV gp120 in healthy volunteers.

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 α4ß7 integrin-binding domain. The breadth of responses induced by this DNA vaccine regimen was comparable to that of viral-vectored vaccine regimens.

A phase I double blind, placebo-controlled, randomized study of a multigenic HIV-1 adenovirus subtype 35 vector vaccine in healthy uninfected adults.

BACKGROUND: We conducted a phase I, randomized, double-blind, placebo-controlled trial to assess the safety and immunogenicity of escalating doses of two recombinant replication defective adenovirus serotype 35 (Ad35) vectors containing gag, reverse transcriptase, integrase and nef (Ad35-GRIN) and env (Ad35-ENV), both derived from HIV-1 subtype A isolates. The trial enrolled 56 healthy HIV-uninfected adults. METHODS: Ad35-GRIN/ENV (Ad35-GRIN and Ad35-ENV mixed in the same vial in equal proportions) or Ad35-GRIN was administered intramuscularly at 0 and 6 months. Participants were randomized to receive either vaccine or placebo (10/4 per group, respectively) within one of four dosage groups: Ad35-GRIN/ENV 2×10(9) (A), 2×10(10) (B), 2×10(11) (C), or Ad35-GRIN 1×10(10) (D) viral particles. RESULTS: No vaccine-related serious adverse event was reported. Reactogenicity events reported were dose-dependent, mostly mild or moderate, some severe in Group C volunteers, all transient and resolving spontaneously. IFN-γ ELISPOT responses to any vaccine antigen were detected in 50, 56, 70 and 90% after the first vaccination, and in 75, 100, 88 and 86% of Groups A-D vaccine recipients after the second vaccination, respectively. The median spot forming cells (SFC) per 10(6) PBMC to any antigen was 78-139 across Groups A-C and 158-174 in Group D, after each of the vaccinations with a maximum of 2991 SFC. Four to five HIV proteins were commonly recognized across all the groups and over multiple timepoints. CD4+ and CD8+ T-cell responses were polyfunctional. Env antibodies were detected in all Group A-C vaccinees and Gag antibodies in most vaccinees after the second immunization. Ad35 neutralizing titers remained low after the second vaccination. CONCLUSION/SIGNIFICANCE: Ad35-GRIN/ENV reactogenicity was dose-related. HIV-specific cellular and humoral responses were seen in the majority of volunteers immunized with Ad35-GRIN/ENV or Ad35-GRIN and increased after the second vaccination. T-cell responses were broad and polyfunctional. TRIAL REGISTRATION: ClinicalTrials.gov NCT00851383.

Measuring human T cell responses in blood and gut samples using qualified methods suitable for evaluation of HIV vaccine candidates in clinical trials.

The next generation of candidate HIV vaccines include replicating vectors selected for tropism to mucosal sites, where an efficacious T cell response will be required to limit T cell replication and HIV associated CD4 T cell loss. To fully assess immunogenicity of such candidates, there is a need to develop robust quality controlled analysis of gut derived HIV specific CD8+ T-cell responses. Despite obvious challenges in obtaining sufficient amounts of tissue, the highly compartmentalised nature of the mucosal immune responses, requires the assessment of CD8 T cells isolated directly from local tissue before any conclusions regarding the induction of mucosal responses are made. Here we describe the optimisation and subsequent qualification of a qualitative and quantitative polychromatic flow cytometry assay to assess antigen specific CD8+ T cells isolated from the gut, using samples from HIV positive and negative volunteers. Internal quality controls monitored over time, combined with the use of quality gating and standard operating procedures were used to demonstrate the generation of robust and reliable data.

In vivo electroporation enhances the immunogenicity of an HIV-1 DNA vaccine candidate in healthy volunteers.

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.

Equivalence of ELISpot assays demonstrated between major HIV network laboratories.

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-γ) 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 (-1.5%, 1.5%) and CEF (-0.4%, 7.8%) responses, were both contained in the pre-specified equivalence margin of interval [-15%, 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.

Phase 1 safety and immunogenicity evaluation of ADVAX, a multigenic, DNA-based clade C/B' HIV-1 candidate vaccine.

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 IFNgamma 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 IFNgamma 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.

Phase 1 safety and immunogenicity evaluation of ADMVA, a multigenic, modified vaccinia Ankara-HIV-1 B'/C candidate vaccine.

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 [1x10(7) (low), 5x10(7) (mid), or 2.5x10(8) 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 IFNgamma 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 IFNgamma 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.

Concordant proficiency in measurement of T-cell immunity in human immunodeficiency virus vaccine clinical trials by peripheral blood mononuclear cell and enzyme-linked immunospot assays in laboratories from three continents.

The gamma interferon (IFN-gamma) 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-gamma 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-gamma ELISPOT responses. These findings also illustrate the ability to standardize the IFN-gamma 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.

Genital human papillomavirus genotypes in northwestern Tanzania.

Using MY09-MY11 PCR and human papillomavirus (HPV) typing by reverse blot hybridization, we found a 34% cervical HPV prevalence among 561 pregnant women in Tanzania. One hundred three of 123 women (84%) with typeable samples harbored high-risk oncogenic strains. HPV type 16 (HPV-16) was the most prevalent subtype (18%) among HPV-infected women and among women with cervical neoplasia (3 of 19). A multivalent vaccine for HPV-16, -18, -31, -33, and -35 would be necessary to prevent 50% of the neoplasia in this population.