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Functional Immune Measurement Across HIV Vaccine and Cure Research
Accelevir provides these assays to support functional immune measurement across HIV vaccine and cure research, including:
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Molecular & Genomic Testing
HIV Reservoir & Virology
Immunology
Sample Processing & Biobanking
Therapeutic Modalities
While a standard ELISA provides the magnitude of antigen-specific humoral immunity, the ELISpot assay provides insight to antigen-specific cellular immunity, and at a higher sensitivity than ELISA.
Accelevir Diagnostics, a CAP-accredited, CLIA-certified bioanalytical laboratory in Baltimore, Maryland, provides HIV-focused ELISpot assay services alongside intracellular cytokine staining and antigen-induced T-cell proliferation, all performed in a single laboratory under one quality system, with qualification documentation available to support regulatory filings.
Baltimore, Maryland · BSL-2+ facility
Accelevir’s ELISpot, ICS, and proliferation assay services are built for the specific challenges of HIV immunology: low-frequency cell responses against highly variable viral antigens, precious sample volumes from clinical trials, and regulatory expectations that demand reproducible, well-documented results.
ELISpot counts how many cells secrete a given cytokine in response to HIV-1 antigen, making it the efficient choice when your endpoint is response magnitude tracked across many participants and timepoints. ELISpot utilizes an antibody capture technique in a 96-well plate: a capture antibody binds secreted proteins (typically IFN-γ) onto a membrane, and a detection antibody paired with an enzyme linked immunosorbent-type chromogenic substrate reveals each cytokine secreting cell as a discrete spot. An automated reader counts spots, normalizing results to spot forming cells (SFC) per 10⁵ or 10⁶ PBMCs.
This workflow is commonly applied in therapeutic vaccine immunogenicity studies, cure-directed analytical treatment interruption trials, and cohort studies comparing immune responses in viremic versus ART-suppressed patients. For instance, ELISpot data have revealed key differences between HIV-1 controllers and progressors in both the magnitude and preservation of T cell effector functions over time as well as been used to evaluate the efficacy of therapeutic vaccination in ART-suppressed individuals aimed at producing long-term, ART-free control of the virus.
ELISpot provides quantitative data while requiring minimal numbers of PBMCs for analysis. The assay can measure responses to up to four cytokines simultaneously, and PBMCs can be analyzed immediately or frozen for later testing. Accelevir’s ELISpot assay services help to standardize and maintain assay consistency in multi-center trials where sample handling varies across sites.
An automated reader counts spots and normalizes results to spot forming cells per 10⁵ or 10⁶ PBMCs.
Multicolor panels resolve which T cell subset responds and whether responses are polyfunctional.
Intracellular cytokine staining extends beyond spot counting to multi-parameter characterization of responding T cells.
This approach resolves questions ELISpot cannot answer: which T cell subset is responding, whether responses are polyfunctional (producing multiple cytokines simultaneously), and what memory phenotype predominates. Beyond cytokine production, ICS panels can be extended to activation and differentiation marker expression to characterize the responding population.
Accelevir’s flow cytometry services support multicolor panels with rigorous compensation, viability gating, and rare-event analysis. With both conventional and spectral flow cytometry instrumentation and expertise, our team can support both targeted ICS panels as well as complex, multiparameter panels combining dozens of cytokines and cell-surface markers for in-depth analysis of cellular responses to antigen stimulation.
Using CFSE or CellTrace dye dilution over 3–7 days of culture, the assay quantifies the percentage of divided cells and stimulation indices in both the CD4 and CD8 compartments.
Why measure expansion capacity rather than effector function alone? Pre-interruption proliferative responses have been reported to track with the duration of virologic control after ART interruption while showing little relationship to how quickly virus first becomes detectable, suggesting that proliferative capacity reflects the ability to sustain control rather than to prevent the initial burst of viremia. Those associations have been specific to the CD8 compartment, which is why resolving CD4 and CD8 expansion separately matters and why a bulk readout can obscure the signal your study is looking for.
In addition, enhanced HIV-specific proliferative capacity has been a consistent feature of long-term non-progressor and post-intervention control phenotypes. And proliferation capacity can change over time. In primary HIV infection studies, CD8 T cell proliferation was measurably impaired during acute infection but recoverable in chronic infection, distinguishing immune reconstitution stages that ELISpot alone does not capture. Importantly, Accelevir pairs proliferation data with phenotyping to identify which T cell memory subsets drive the expansion.
The antigen you stimulate against shapes what you see. Responses directed against the highly conserved Gag protein have been associated with virologic control more consistently than responses to Nef, Pol, or Env, and Nef- and Pol-specific responses decline during the first year of ART, so participants on long-term therapy may show little of either. Antigen panel selection is therefore a study design decision rather than a default, and one worth making before your samples are collected.
Dye dilution over 3–7 days of culture quantifies divided cells and stimulation indices.
These three assays are most informative together, and the reason is not simply that more data is better.
Each captures a different property of the same response, and the combinations carry information the individual assays do not. Cytokine secretion and proliferative capacity are correlated but not interchangeable, and participants can separate on one while appearing similar on the other, so stratifying on both has been reported to identify sustained virologic control more effectively than either alone.
ICS then explains what ELISpot and proliferation measured: whether a response resides in the CD4 or CD8 compartment, which memory subset is expanding, and whether responding cells are polyfunctional. As such, a combination approach is essential to a program seeking to define whether an intervention improved the quality of an immune response rather than only its size.
Because all three require the same sample type, can employ the same peptide pools and controls, and are performed in the same laboratory, you can scope them against a single collection and interpret them against one another rather than across vendors.
Your study team and Accelevir scientists jointly define antigen pools, cytokine targets, positive control stimuli (SEB, PHA, or study-specific controls), and negative controls. Key reagents must be validated for assay setup, including lot-specific qualification of capture antibody, detection antibody, and membrane plates. Peptide pool selection accounts for HIV-1 subtype representation, peptide length for CD4 versus CD8 detection, and epitope coverage for determinant mapping. Pools are commercially sourced or client-provided, and we can qualify custom panels for use in these assays.
Before clinical sample analysis begins, Accelevir runs qualification experiments assessing specificity, sensitivity (limit of detection and limit of quantitation), precision, and robustness. Qualification includes assessing intra-assay, inter-assay, and inter-analyst precision using characterized PBMC reference lots. Cell-based functional assays can exhibit higher variability than ligand binding assays, particularly in low-response samples; our qualification protocols account for this by defining acceptance criteria calibrated to the biological variability of each analyte and antigen combination.
Clinical samples are processed under chain-of-custody documentation with controlled storage conditions. Results include raw spot counts, background-subtracted SFC per million PBMCs, replicate well concordance, positive control acceptance, gating strategies (for ICS and proliferation), and analysis against defined positivity cutpoints.
Scope your immune monitoring endpoints with our team.
Because all three assays use the same sample type and laboratory, they can be scoped against a single collection.
Accelevir has qualified overlapping peptide pools covering HIV-1 Gag, Pol, Env, and Nef proteins. Standard pools use 15–20mer peptides with 10–11 amino acid overlap for combined CD4 and CD8 T cell detection. Shorter 9–11mer optimal epitope peptides are available for MHC-I-restricted CD8 responses. Pools are commercially sourced or client-provided. For studies requiring coverage of specific HIV-1 subtypes, accessory proteins, or novel immunogens, we can qualify custom panels for use in these assays.
These assays require high-quality cells for reliable results. Accelevir’s PBMC processing services include controlled-rate cryopreservation, validated thaw protocols, and viability qualification before assay setup. PBMCs can be analyzed immediately or frozen for later testing. All samples remain under documented chain of custody with temperature-monitored storage. For limiting samples, our low-input workflows are optimized to maximize data from minimal cell numbers.
Accelevir operates a CAP-accredited, CLIA-certified, high-complexity laboratory. ELISpot, ICS, and proliferation assays are analytically qualified and performed under a unified quality management system, with qualification documentation available to support regulatory filings. Our protocols include lot-qualified reagents, replicate wells, defined acceptance criteria for positive control performance, and defined cutpoints for calling positive responses.
Yes. We can qualify commercially sourced or client-provided peptide pools for use in these assays, and can accommodate alternative cytokine readouts or non-standard stimulation conditions. Timelines depend on antigen complexity and qualification scope; typical custom panel qualification runs 4–8 weeks from reagent receipt through report delivery. Custom panels can target epitopes identified through determinant mapping or sequence-based prediction.