AIDS Vaccination Studies with an Ex Vivo Feline Immunodeficiency Virus Model: Analysis of the Accessory ORF-A Protein and DNA as Protective Immunogens

Abstract

Determining which antigen must be included in AIDS vaccines to confer maximum protection is of utmost importance. In primate models, vaccines consisting of or including accessory viral proteins have yielded conflicting results. We investigated the protective potential of the accessory protein ORF-A of feline immunodeficiency virus (FIV) in cats. All three immunization strategies used (protein alone in alum adjuvant, DNA alone, or DNA prime-protein boost) clearly generated detectable immune responses. Upon challenge with ex vivo homologous FIV, ORF-A-immunized cats showed distinct enhancement of acute-phase infection relative to mock-immunized animals given alum or empty vector DNA. This effect was tentatively attributed to increased expression of the FIV receptor CD134 that was observed in the immunized cats. However, at subsequent sampling points that were continued for up to 10 months postchallenge, the average plasma viral loads of the ORF-A-immunized animals were slightly but consistently reduced relative to those of the control animals. In addition, CD4؉ T lymphocytes in the circulation system declined more slowly in immunized animals than in control animals. These findings support the contention that immunization with lentiviral accessory proteins can improve the host's ability to control virus replication and slow down disease progression but also draw attention to the fact that even simple immunogens that eventually contribute to protective activity can transiently exacerbate subsequent lentiviral infections.

Env-Expressing Autologous T Lymphocytes Induce Neutralizing Antibody and Afford Marked Protection against Feline Immunodeficiency Virus

“…Humoral immunity was assessed at the start of immunization, when all animals were uniformly negative (data not shown), at boost, and at challenge. In line with data from previously reported FIV DNA vaccination studies (24,25,37,54), at boost, serology was still essentially negative except for two vaccinees (cats GD and GE) who, somewhat surprisingly, showed measurable anti-Env binding antibodies and NA. On the other hand, at challenge, all vaccinees possessed moderate to high levels of anti-whole-FIV antibodies and anti-Env binding antibodies, and, most impor- (Table 1).…”

“…CTL activity in unstimulated PBMCs was measured with a fluorescence-based assay using Env-and GFP-transduced immortalized autologous fibroblasts as targets (5). For the enumeration of IFN-␥-secreting T lymphocytes, PBMCs were restimulated in vitro with a pool of 15-mer peptides encompassing the entire Env and directly examined by an enzyme-linked immunospot (ELISpot) assay (54). For clarity, only measurements above the cutoff are shown.…”

“…Samples were analyzed in triplicate by using CELLQuest, version 2, software, and the percent specific anti-Env CTL activity was calculated by using the following formula: ( Env-specific IFN-␥-secreting T lymphocytes. Gamma interferon (IFN-␥)-secreting T lymphocytes were counted by an enzyme-linked immunospot (ELISpot) assay using pooled 15-mer peptides overlapping the entire Env of FIV-Pet as an antigen and the feline IFN-␥ development module (R&D Systems, Minneapolis, MN) (54). Briefly, 100 l of IFN-␥ capture antibody was poured into 96-well polyvinylidene difluoride (PVDF) microplates (Millipore, Milan, Italy) and kept overnight at 4°C in the dark.…”

Ferritin as a reporter gene for in vivo tracking of stem cells by 1.5-T cardiac MRI in a rat model of myocardial infarction

“…The supernatant of transfected 293-T cells was collected 48 -72 h post-transfection, clarified by centrifugation, filtered, and stored at Ϫ80°C in small aliquots until use. LAW34-GFP, LAW34-BRCA1, and LAW34-hFTH pseudotyped particles were quantified by testing reverse transcriptase activity (40) and titrated in 293-T cells by flow cytometry (40). Titers were expressed as number of transduction units (TUs) per milliliter.…”

A Lentiviral Vector-Based, Herpes Simplex Virus 1 (HSV-1) Glycoprotein B Vaccine Affords Cross-Protection against HSV-1 and HSV-2 Genital Infections

“…To check for immune cell depletion, mice were bled the following day retro-orbitally; circulating CD4 ϩ and CD8 ϩ T lymphocytes were labeled with fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies (eBioscience, San Diego, CA) and counted using flow cytometry as described previously (42). gB1 expression.…”