Utilization of Feline ELISPOT for Mapping Vaccine Epitopes Protocol

Overview

Introduction of Feline ELISPOT Assay

Numerous human immunodeficiency virus (HIV)/AIDS researchers have used the ELISPOT analysis system to evaluate the epitopes on the human immunodeficiency virus-1 (HIV-1) viruses that induce mediators of cell-mediated immunity (CMI), such as interferon-J (IFNγ), IFND, IL-2, tumor necrosis factor-D (TNFD), and cytolytic mediators (e.g., perforins and granzymes). The most widely used ELISPOT studies in HIV/feline immunodeficiency virus (FIV) research detect the cytokines IFNγ and IL-2. IFNγ and IL-2 ELISPOT assays are among the initial ELISPOT systems to be commercially available for both humans and cats. IFNγ is an important mediator of cellular immunity expressed by CD4⁺ T-helper 1 (Th1) cells, NK cells, and cytotoxic T lymphocytes (CTLs). IL-2 is produced by T cells (both CD4⁺ and CD8⁺ T cells), but the predominant T-cell population that produces this cytokine is the Th1 cells. The general function of IL-2 is to promote the proliferation and differentiation of effector cells, such as T cells, NK cells, and even B cells, as well as autocrine effects promoting proliferation of Th cells. The generation of FIV- or HIV-specific antibodies is also mediated by cytokines, such as IFNγ and IL-2. Peripheral blood mononuclear cells (PBMCs) from HIV-1-infected subjects and FIV-infected cats typically express IFNγ after stimulation with HIV-1 and FIV peptides, respectively. More recently, HIV-specific IL-2 responses of T cells alone or in combination with IFNγ responses correlated with the decreased HIV-1 load in infected individuals. Studies are being performed on infected hosts with an expectation that the immunity for controlling infection may be utilized in the immunity for vaccine prophylaxis. However, controversies exist on whether IFNγ is involved in vaccine protection against these viruses. Taking a conservative viewpoint, effective FIV and HIV-1 vaccines need to generate both robust CTL activity and virus-neutralizing antibodies (VNAs) against the respective viruses. Thus, because IL-2 and IFNγ play a critical role in both humoral and cellular immunities, they are important cytokines to monitor during AIDS lentivirus infection as well as in vaccine responses to be leveraged for vaccine development.

The ELISPOT system is a powerful screening tool that allows for the identification of functional T-cell responses to various antigens, including whole organisms, proteins, and peptides. The use of protein-derived, overlapping peptide antigens within the ELISPOT system facilitates the identification of peptide-specific epitopes based on the peptide-specific immune responses generated by the PBMC. In the culture milieu of the ELISPOT well, T cells recognize peptides presented by major histocompatibility complex (MHC), which in turn triggers the T cells to express CMI mediators, such as IL-2 and IFNγ. Sufficient levels of CMI mediator expressed during short-term antigen stimulation surrounding an individual cell are captured by the mediator-specific antibodies on the ELISPOT membrane and detected by a chemical reaction similar to that of ELISA. A unique aspect of the ELISPOT system is the one-to-one relation between the mediator-expressing cell and the “spot” resulting from such reaction. The direct use of purified cell populations, such as CD4⁺ T cells and CD8⁺ T cells, to the ELISPOT system has aided in identifying the cell type(s) expressing the CMI mediators. In the case of IFNγ, this step determines the numbers of CD4⁺ Th1 cells, CD4⁺ CTL, or CD8⁺ CTL producing the IFNγ in response to stimulation of specific epitopes.

IFNγ and IL-2 ELISPOT assays are being used to identify and map specific epitope(s) on individual viral peptides that stimulate these cytokine productions by the T cells from vaccinated animals and humans. The sizes of the peptides generally used for initial screening are those that can generate both MHC-I- and MHC-II-mediated responses. MHC-I presents small peptides of 8–11 amino acid (aa) to the T-cell receptor (TCR) of CD8⁺ T cells while MHC-II presents large peptides of 10–30 aa to TCR of CD4⁺ T cells. Thus, peptides of about 15 aa are typically used to generate both MHC-I and -II presentations of a peptide to CD8⁺ T cells and CD4⁺ T cells, respectively. To assure that all epitopes on the full length of the targeted viral protein are evaluated, overlapping 15-mer peptide with 8–11 aa overlaps is generated using a computational algorithm that generates peptide sequences based on the rules for CTL epitopes, such as the PeptGen Peptide Generator tool. Pools of peptides can be used as a first screening tool to identify regions of the protein that initiate a positive mediator response. Following the use of pooled peptides, individual peptides can be used to further localize epitopes. Upon detection of positive mediator response, the specific epitope(s) on the 15-mer peptide are identified for the MHC-I-mediated responses by testing with smaller peptide sequences, whereas the exact peptide size for the MHC-II-mediated response can be determined by using different sequence overlaps of the peptide. Candidate CTL epitopes in combination with Th1 epitopes are being used as an HIV vaccine immunogen in multipeptide complexes. This vaccine approach can be tested against lentivirus challenge, utilizing animal models, such as FIV vaccine in cats. To achieve this goal, IFNγ and IL-2 ELISPOT systems of humans and cats are being applied.

Materials of Feline ELISPOT Assay

FIV Vaccines

Prototype dual-subtype FIV vaccine (hereon called prototype IWV or IWV vaccine) consists of 250 Pg each of 0.1% paraformaldehyde inactivated FIV_Pet and FIV_Shi viruses (IWV) mixed in 1 mL of FD-1 adjuvant (Fort Dodge Animal Health, Fort Dodge, IA) and supplemented with human or feline IL-12 at 5 Pg per dose.
Commercial dual-subtype FIV vaccine called Fel-O-Vax^® FIV (Fort Dodge Animal Heath) was purchased from Veterinary Medicine Teaching Hospital at the University of Florida. This vaccine consists of 2 × 107 cells/dose of inactivated FIV_Pet infected cells and inactivated FIV_Shi-infected cells and d50 Pg/ dose of inactivated whole viruses of FIV_Pet and FIV_Shi.

Peripheral Blood Mononuclear Cell Preparation and T Cell Isolation

Ficoll-hypaque.
Hanks’ balanced salt solution (HBSS) with 5% EDTA.
Dynabeads^® Untouched™ Human CD4⁺ T Cells (Invitrogen, Oslo, Norway) for isolation of CD4⁺ T cells. This kit includes Depletion MyOne™ Dynabeads and antibody mix (human CD4⁺ T cells).
Dynabeads^® Untouched™ Human CD8⁺ T Cells (Invitrogen, Oslo, Norway) for isolation of CD8⁺ T cells. This kit includes Depletion MyOne™ SA Dynabeads and antibody mix (human CD8⁺ T cells).
Isolation buffer: Ca²⁺- and Mg²⁺-free phosphate-buffered saline (PBS) supplemented with 0.1% bovine serum albumin (BSA) and 2 mM EDTA.

IFNg and IL-2 ELISPOT for Humans and Cats

96-Well polyvinylidene fluoride (PVDF)-membrane plates (Millipore MultiScreen plates, Millipore, Billerica, MA).
AIM-V^® medium supplemented with gentamycin (25 Pg/mL) and with either 10% heat-inactivated pooled HIV seronegative human sera or 10% heat-inactivated pooled specific-pathogen-free (SPF) feline sera for use with the human or feline PBMC, respectively (see Note 1).
PBS –137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2 HPO₄, 1.5 mM KH₂PO₄, pH 7.4.
Reagent diluent: PBS supplemented with 1% (w/v) fraction V BSA for use in reconstituting lyophilized antibodies (see Note 1).
Blocking buffer – 1% BSA, 5% (w/v) sucrose in PBS (see Note 1).
FeIFNγ ELISPOT Development Module. The development module includes a matched pair of antibodies used for capture and detection:

Feline IFNγ Capture Antibody Concentrate reconstituted in 1 mL PBS and diluted 1 in 50 parts reagent diluent.
Feline IFNγ Detection Antibody Concentrate reconstituted in 1 mL PBS and diluted 1 in 50 parts reagent diluent.

FeIL-2 ELISPOT Development Module. The development module includes a matched pair of antibodies used for capture and detection:

Feline IL-2 Capture Antibody Concentrate reconstituted in 1 mL PBS and diluted 1 in 50 parts reagent diluent.
Feline IL-2 Detection Antibody Concentrate reconstituted in 1 mL PBS and diluted 1 in 50 parts reagent diluent.

ELISPOT Blue Color Module or equivalent: The color module contains the following:

Streptavidin–AP concentrate (streptavidin conjugated to alkaline phosphatase) diluted 1 in 50 parts with Reagent Diluent (see Note 2).
5-bromo-4-chloro-3c indolylphosphate p-toluidine salt (BCIP)/nitro blue tetrazolium (NBT) chromogen consists of BCIP and NBT chloride in organic solvent (see Note 2).

Human IFNγ ELISPOT Kit and Human IFNγ ELISPOT Development Module. Both MABTECH and R&D kits contain a matched pair of antibodies to be used for capture and detection, but only MABTECH kit includes streptavidin–AP solution and BCIP/NBT chromogen for detection.

Antigens

Overlapping 15-mer peptides diluted in AIM-V^® medium: The overlapping peptides were derived from the following proteins and synthesized at the respective laboratories.

HIV-1_HXB2 subtype-B p24.
FIV_Bang.

Concanavalin A (ConA) diluted in AIM-V^® medium for use as a positive control.
Recombinant HIV-1 and FIV p24 proteins and FIV IWV are diluted in AIM-V^® medium for use as positive controls.

Methods of Feline ELISPOT Assay

Two strategies using the FIV–cat model have been undertaken to identify the vaccine epitopes essential for developing an effective HIV-1 vaccine for humans. The first approach is to identify the vaccine epitopes and the specific immune responses induced by the prototype and commercial FIV vaccines. These vaccines have been shown to confer protection against global FIV subtypes and recombinants. This approach is based on the concept that the overall humoral and cell-mediated immune responses, including the epitopes that generate appropriate protective immune responses determined from this model, can translate to an effective HIV-1 vaccine for humans. The counterpart HIV-1 epitopes are anticipated to induce similar protective immune responses. The first step toward analyzing the protective vaccine immunity is to measure the magnitude of the immune responses stimulated by the vaccine. Vaccine-induced VNAs to FIV have recently been evaluated for both prototype and commercial FIV vaccines. In these studies, vaccine-induced VNAs conferred protection against viruses with similar envelope (Env) sequences, but not against viruses with distinctly heterologous Env sequences, such as those from subtypes different from the vaccine viruses. These findings suggest that protection against heterologous subtype viruses and recombinant viruses likely require a robust T-cell immunity to multiple viral proteins.

The induction of FIV-specific T-cell immunity has been reported for only prototype FIV vaccine using both mRNA and biological tests to detect T-cell functions. The mRNA assays for T-cell cytokines and cytolytic mediators are an indirect approach and do not always correspond with the protein levels of these molecules. Biological assays are ideal, but many are impractical for analyzing a multitude of epitopes on the viral proteins. The current trend is to combine epitopes identified by in silico immunoinformatics to be later tested with a rapid biological confirmatory system, such as ELISPOT. Since IFNγ and IL-2 mRNA analyses, FACS intracellular staining (ICS) for IFNγ, and proliferation assay for IL-2 detected robust IFNγ and IL-2 expressions by the PBMC from prototype FIV-vaccinated cats, the next approach was to determine the viral epitopes responsible for vaccine protection. HIV-1 core p24 and reverse transcriptase (RT) have a large number of CTL epitopes according to LANL database. The viral protein counterparts of HIV-1 also exist for FIV. However, counterparts for a few HIV-1 regulatory proteins do not exist for FIV, such as HIV Nef. Thus, T-cell epitopes on FIV p24 and RT were screened by IFNγ ELISPOT to (1) determine whether the commercial dual-subtype FIV-infected cell vaccine can induce potent cellular immunity as described for prototype dual-subtype IWV vaccine, (2) identify potential CTL epitopes on viral proteins that are recognized by the T cells from vaccinated cats, and evaluate whether FIV p24-specific IFNγ responses of PBMC correlates with IL-2 responses.

The second approach is to identify evolutionarily conserved vaccine epitopes on HIV-1 and FIV that confer protection against these viruses. This approach is founded on a concept that conserved regions are present on both viruses to maintain either the structural or the functional stability of the virus, which is less likely to be mutable and more likely to be broadly conserved among all subtypes of the viruses. In support of this approach, recent studies demonstrate cross-protection of HIV-1 p24-immunized cats against low-dose FIV challenge. Additional studies show that PBMC from HIV-infected human subjects and PBMC from HIV-1 p24-vaccinated cats are stimulated by similar HIV-1 p24 peptides and most remarkably to a number of FIV p24 peptides. Interestingly, these are similar FIV p24 peptides recognized by the prototype IWV-vaccinated cats. Preliminary results suggest that PBMCs from HIV-infected subjects also respond to conserved FIV RT peptide pools. Conserved epitopes on these viral proteins are currently being assessed by phenotype/function-based ICS and ELISPOT analyses for their ability to induce CTL activity. These techniques in combination with MHC-based immunoinformatics expedite the identification of CTL and Th epitopes essential for formulating the vaccine immunogen against HIV-1 and FIV. More importantly, such techniques are also being used to characterize vaccine epitopes for a wide variety of pathogens.

Human and Animal Subjects

SPF cats were vaccinated 2× to 4× with Fel-O-Vax^® FIV or prototype IWV at intervals of 3–4 weeks. In one study, SPF cats were vaccinated 7× to 9× over the course of 4 years initially with IWV vaccine and later on occasionally in combi nation with Fel-O-Vax^® FIV. Since the initial four vaccinations in the first year used only IWV vaccine and all vaccinations subsequently contained IWV vaccine. Blood samples were collected in heparin at 1–3 weeks after the third or fourth vaccination and used fresh.
HIV-1 positive Caucasian male subjects from the University of California at San Francisco (UCSF) cohort study were used in current studies. These subjects consisted of 12 HIV-1-infected long-term survivors who were not on antiretroviral therapy (ART), an HIV-1 long-time-infected subject who was on ART, and an HIV-1 short-time-infected subject who was not on ART. Blood samples were collected in heparin and used fresh.
All human studies were approved by the Committee on Human Research at the UCSF and at the University of Florida. All animal studies were approved by the Institutional Animal Care and Use Committee at the University of Florida.

Isolation of Peripheral Blood Mononuclear Cells

The human PBMCs are isolated from blood by Ficoll-hypaque-based gradient purification method as previously described. The CD4⁺ and CD8⁺ T-cell populations are obtained from PBMC by positive selection using magnetic beads bearing anti-human CD4 monoclonal antibodies or anti-human CD8 monoclonal antibodies according to the manufacturer’s instruction (see Note 3).

Antigen Preparation

Overlapping peptides based on the p24 protein of FIV and HIV are derived by using PeptGen Peptide Generator tool, which generates peptide sequences based on the rules for CTL epitopes as previously described. This peptide generator tool is used to construct overlapping 15-mer peptide sequences with 11 aa overlaps for HIV-1 p24. The FIV p24 peptide pools were generally 15-aa peptide (median of 15 aas and range of 12–17 aas) with 11 aa overlap (median of 11 aas and range of 7–12 aa overlaps).
With the exception of peptide-pool Hp1 consisting of four overlapping peptides from the amino-terminal, each peptide pool from amino-end Hp2 to carboxyl-end Hp18 contains three consecutive overlapping peptides. The three 15-mer peptides in each peptide pool have the peptide designation of HCP# for HIV-1 Core p24 Peptide number.
Peptides used to stimulate PBMC are used at a concentration of 2 Pg peptide each per well for pooled peptide analysis and 5 Pg peptide per well for individual peptide analysis.
Positive controls, recombinant HIV-1_LAV p24 (identical in p24 sequence to HIV-1_HXB2) and recombinant subtype-A FIV_Pet, and FIV_Bang p24 proteins are expressed in E. coli M15 and purified as previously described.
Recombinant FIV and HIV p24 proteins are used to stimulate PBMC at concentrations of 2 μg protein per well.
FIV IWV (same as vaccine immunogen, but inactivated with UV and heat) is used to stimulate PBMC at 2 μg per well.
T-cell mitogen, Con A, is used to stimulate PBMC at 2.5 μg per well.
FIV_Bang p24 10-mer peptide (VGSPGYKMQLL) is used as a negative control for HIV-1 p24 peptides as this peptide had little to no IFNγ response with PBMC from HIV-1 p24- immunized cats and HIV-seronegative subjects.
Lipopolysaccharide (LPS) from E. coli M15 is used as a control for both recombinant HIV-1LAV and FIV p24 proteins. E. coli LPS is used at 5 and 50 EU per well.

Feline and Human IFNγ ELISPOT Analyses

96-well PVDF membrane plates are primed by adding 30 μL of 35% ethanol in each well and incubating at room temperature for 60 s.
The wells are washed 3× with PBS (250 μL per well) using a squirt bottle, multichannel pipette, or plate washer. Remove any remaining PBS by inverting the plate and blotting it against a clean paper towel (see Note 4). A plate washer can also be used if available.

Figure 1. IFNJ responses to FIV and HIV-1 p24 and reverse transcriptase (RT) peptide pools by HIV-1-infected subjects.

Once the plates are primed, immediately (see Note 5) add 100 μL of Capture Antibody Solution at 1:50 dilution to each well, cover, and incubate for at least 10 h at 2–8°C.
Aspirate Capture Antibody Solution from each well and wash 3× with PBS (250 μL per well). After the final wash, remove any remaining liquid by inverting the plate and blotting it against a clean paper towel (see Note 5).
Block the membranes by adding 250 PL of AIM-V^® medium with gentamycin and 10% serum (either feline or human) to each well. Incubate for 2 h at 37°C.
Aspirate blocking medium: Wash 3× with PBS (250 μL per well) and tapping-dry the plates. Rinse wells with AIM-V^® medium with gentamycin.
AIM-V^® medium with gentamycin (50 μL) is added to each well. Incubate at room temperature until addition of antigen and PBMC.
Antigen (peptide, peptide pool, recombinant protein, or controls) or medium diluted only in AIM-V^® medium with gentamycin (50 μL) is added to each well (see Note 6).
PBMC (100 PL) is added at a concentration of 1 × 106 to 5 × 106 cells per mL diluted in AIM-V^® medium with 10% serum (either feline or human) (see Note 7).
Incubate the covered plates for 24 to 36 h at 37°C in 5% CO2 (see Note 8).
The wells are washed 5× with PBS (250 μL per well). After the final wash, remove any remaining liquid by inverting the plate and blotting it against a clean paper towel.
Detection Antibody Solution (100 μL) is added at 1:50 dilution to each well and incubated for 12 h or overnight at 2–8°C.
The wells are washed 5× with PBS (250 μL per well). After the final wash, remove any remaining liquid by inverting the plate and blotting it against a clean paper towel.
Streptavidin–AP solution (100 μL) is added to each well and incubated for 2 h at room temperature.
Wash the wells 5× with PBS (250 μL per well). After the final wash, remove any remaining liquid by inverting the plate and blotting it against a clean paper towel.
BCIP/NBT chromogen solution (100 μL) is added to each well, covered, and incubated for 45–60 min at room temperature in the dark.
Rinse with deionized water. Invert plate and tap to remove excess water. Allow the plate to dry at room temperature or at 37°C (approximately 2 h).
Once the plates are fully developed, the spots are quantitated either manually using a dissecting microscope or using an automatic spot counter and photographs taken (see Note 9).
Following quantification, the number of spots in the negative controls is subtracted from all sample wells to determine the spot-forming units (SFUs) (see Note 10) and typically reported as SFU per 10⁶ cells. Greater than 50 SFU per 10⁶ PBMC is considered significant.
Peptide pools that return positive results are then retested as individual peptides.

Notes of Feline ELISPOT Assay

These reagents should be filter sterilized with a 0.2-P m filter before use.
These reagents should be stored at 2–8°C with caution not to freeze.
The purity of the magnetic-bead purified human CD8⁺ T-cell population in our study ranged from 69 to 88% CD3⁺ CD8⁺ T cells with 0.3–10% CD3⁺ CD4⁺ T cells, and the human CD4⁺ T-cell population was 90–92% CD3⁺ CD4⁺ T cells with 1–10% CD3⁺ CD8⁺ T cells.
Do not dry the plates before the Capture Antibody Solution is ready.
Do not touch the membranes during washing to avoid damage.
The concentration of the working solutions for each stimulant is fourfold higher than the final concentration in the well.
The final volume after addition of antigen and PBMC is 200 PL per well, and the cell concentration is 0.1 × 10⁶ to 0.5 × 10⁶ cells per well.
Care should be taken not to move, bump, or expose plates to vibrations that may cause the cells to move, as the assay is dependent on cells remaining in place to create a “spot.”
The uses of the dissecting microscope and the automatic reader both have pros and cons. With the dissecting microscope, the investigator has more individual and controlled input regarding what is deemed a “spot,” making it easier to sort out artifactual marks and dealing with varying background intensities that are common in ELISPOT. However, because the determination is subjective, consistency can be difficult. For example, in wells with few spots, small pale spots are more obvious than in wells with numerous intense spots. In addition, manual counting is extremely labor intensive. Automatic readers are quick (approximately 4 min per 96-well plate) and are able to read each well with the same thresholds and algorithms enhancing consistency. When using an automatic reader, it is imperative to set thresholds and spot check spot numbers for each plate or assay to avoid underestimating or overestimating spots. Automatic readers often have problems with artifacts if threads or dust get in the wells; however, built-in algorithms attempt to detect gradients in the spots to distinguish between artifacts and true “spots” as well as subdividing coalescing spots.
SFUs are considered to each represent a single cell secreting IFNγ. Although not typically considered in the current literature, the size and intensity of the spots may add additional information regarding the level of IFNγ secretion as well as the number of secreting cells. The SFU in positive controls especially often coalesces and makes enumerating individual spots difficult or impossible. In these cases, the percent saturation of the well (e.g., 80%) can be used as a measurement (some automatic readers generate this data).