HIV/AIDS

Vaccine Assessment

Developing a safe and effective vaccine requires laboratories analyzing samples from clinical trials to follow good clinical laboratory practices (GCLP). The laboratories use sensitive, accurate, reproducible, and quantitative assays that provide clear measurements of vaccine-induced immune responses to allow comparison of data across multiple sites, and prioritization of the most promising vaccines. To define the correlates of protection, new and innovative approaches will be required to correlate immune response with vaccine-induced protection. The central components of vaccine assessment include

• Safety Laboratories
  All phases of clinical trials of a candidate HIV vaccine safety testing and monitoring help determine how well the volunteers tolerate the vaccine. Routine safety tests include blood and urine sample analyses that check blood cell counts, hemoglobin levels, kidney fucntion, and liver function; as well as screening for infection with syphilis, HIV, and/or hepatitis B and C viruses.  These tests are performed by laboratories that meet Clinical Laboratory Improvements Amendments (CLIA) certification and accreditation by College of American pathologists (CAP), standards which are normative for all clinical laboratories in the United States. Similarly, international laboratories performing assays for HIV vaccine trials meet local accreditation and are enrolled in CAP and in external quality assurance programs to ensure performance standards.

• PBMC Processing Laboratories
  To ensure the highest possible specimen quality among different clinical settings, optimal methods are required to collect, process, ship, and store blood specimens to ensure appropriate blood cell recovery, viability and function. Site-associated laboratories separate and cryopreserve serum and peripheral blood mononuclear cells (PBMC) from blood within 6 to 8 hours after collection by venipuncture. Appropriate sample volumes are optimally handled to perform immunological evaluations that indicate immune resistance to HIV infection.

• Endpoint Laboratories
  The immunological objective of HIV vaccines is to elicit effective humoral, cellular, and mucosal immune responses. To assess the effectiveness of the new generation of vaccines, scientists have expended considerable efforts into developing and standardizing reliable assays that analyze and quantify vaccine-induced immune responses. Specimens from vaccine clinical trials—PBMCs and serum or plasma—are currently evaluated using four optimized and validated assays of immune response to the vaccine. The assays are performed at designated "central" laboratories under internationally accepted GCLP guidelines. The central laboratories also follow approved Quality Assurance (QA) and Quality Control (QC) protocols and have Standard Operating Procedures (SOP) for the assays. These labs participate in external proficiency testing administered by CAP and DAIDS as a means to ensure confidence in the quality of results, assess assay proficiency, and contribute to the standardization of assay performance among laboratories. The four validated assays include:
  1. IFN-g ELISPOT assay—This assay quantifies the number of fluorescent spots of solid phase-captured interferon gamma (IFN-g) secreted by individual T cells after stimulation with HIV antigens or peptides. 
  2. Intracellular Cytokine Staining (ICS) assay—This assay uses flow cytometry to separate out T cells from other lymphocytes based on selective expression of surface markers and cytokine secretion upon stimulation with HIV antigens or peptides and after cell activation. 
  3. Virus Neutralization Assay—This in vitro assay measures the neutralizing immunoglobulin concentration (titer) in serum or plasma that blocks HIV infectivity prior to entry into target cells. 
  4. ELISA binding antibodies—This assay detects the presence of binding anti-HIV immunoglobulins in patient’s blood, serum, or plasma.

DAIDS has established an external quality assurance (EQA) program for laboratories performing immunogenicity assays as part of NIAID sponsored clinical trials. The EQA program for ELISpot and ICS assays is described below, and programs for other testing such as viral suppression, proliferation, and virus neutralization are in development. Participation in these programs for protocol-mandated assays is a DAIDS requirement.

1. ELISpot EQA Program—Laboratories participating in the EQA program for ELISpot receive and test proficiency panels consisting of donor PBMC samples selected to provide a range of reactivity to stimulatory peptides such as CMV. Testing results are evaluated and compared to determine variability in results between different laboratories. A report is then issued to each participating laboratory that describes intra- and inter-laboratory assay proficiency.
2. Cytokine Flow Cytometry (CFC) EQA Program—Laboratories performing ICS assays participate in the CFC EQA program. Each laboratory is provided a standardized protocol with all materials and reagents. Cytokine responses are measured in CD4+ and CD8+ T-cells to peptides using cryopreserved PBMC from 3 donors previously characterized as low, medium and high responders to a CMV peptide mix. Results from all laboratories are analyzed and a report is generated to provide each participating laboratory the information to assess their performance and to compare their data to that from other laboratories. 

New Technology/New Assays
In addition to the validated assays, there is a need to incorporate new technology platforms into clinical trials of candidate HIV vaccines to comprehensively characterize vaccine-induced responses and to define the correlates of immune protection in large-scale efficacy trials. To accommodate these aims, additional assays currently under development include

• Identification of activated memory CD4 lymphocytes by flow cytometry 
• Proliferative capacity of lymphocytes by flow cytometry

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