James K. Coleman

Advances in FIV vaccine technology

Advances in vaccine technology are occurring in the molecular techniques used to develop vaccines and in the assessment of vaccine efficacy, allowing more complete characterization of vaccine-induced immunity correlating to protection. FIV vaccine development has closely mirrored and occasionally surpassed the development of HIV-1 vaccine, leading to first licensed technology. This review will discuss technological advances in vaccine designs, challenge infection assessment, and characterization of vaccine immunity in the context of the protection detected with prototype and commercial dual-subtype FIV vaccines and in relation to HIV-1.

Feline immunodeficiency virus model for designing HIV/AIDS vaccines.

Feline immunodeficiency virus (FIV) discovered in 1986 is a lentivirus that causes AIDS in domestic cats. FIV is classified into five subtypes (A-E), and all subtypes and circulating intersubtype recombinants have been identified throughout the world. A commercial FIV vaccine, consisting of inactivated subtype-A and -D viruses (Fel-O-Vax FIV, Fort Dodge Animal Health), was released in the United States in 2002. The United States Department of Agriculture approved the commercial release of Fel-O-Vax FIV based on two efficacy trials using 105 laboratory cats and a major safety trial performed on 689 pet cats. The prototype and commercial FIV vaccines had broad prophylactic efficacy against global FIV subtypes and circulating intersubtype recombinants. The mechanisms of cross-subtype efficacy are attributed to FIV-specific T-cell immunity. Findings from these studies are being used to define the prophylactic epitopes needed for an HIV-1 vaccine for humans.

HIV-1 p24 vaccine protects cats against feline immunodeficiency virus infection.

Background:Based on previous analysis of feline immunodeficiency virus (FIV)-specific cross-reactive antibodies to HIV-1 p24, cats vaccinated with HIV-1 p24 were evaluated for cross-reactive immunity to FIV. Objective:To determine the level of cross-reactivity that exists between HIV-1 and FIV p24 and its implications for vaccine prophylaxis. Methods:Specific-pathogen-free cats were immunized three times with HIV-1 p24 in Ribi adjuvant, with (n = 18) or without cytokine (n = 6). Control cats were immunized three times with adjuvant (n = 10) or phosphate-buffered saline (PBS; n = 5). All immunized cats were challenged with either subtypes B or A/B FIV, and monitored by virus isolation, proviral PCR, FIV-specific antibodies, and feline interferon-γ ELISpot for T-cell activities. Results:Of 18 cats vaccinated with subtype B HIV-1 (HIV-1LAI/LAV, HIV-1UCD1) p24 in Ribi/cytokine adjuvant 14 (78%) were protected against FIV challenges (subtype Agag and Bgag) that infected all 15 adjuvant- or PBS-immunized cats. Furthermore, only three of six (50%) cats vaccinated with FIV p24 in Ribi/cytokine adjuvant were protected against similar FIV challenge. HIV-1 p24 vaccination induced weak cross-reactive antibodies to FIV p24, which did not correlate with vaccine efficacy. However, the peripheral blood mononuclear cells from HIV-1 p24-vaccinated/protected cats at 33–34 weeks post-FIV challenge responded to three T-cell responsive peptides at the carboxyl-terminus of the FIV p24, whereas those cells from the infected control cats had minimal to no responses to the same peptides. Conclusions:These results suggest the importance of including lentiviral p24 as vaccine immunogen for human AIDS vaccine. Moreover, these results suggest the potential importance of evolutionarily conserved, cross-protective epitopes in vaccine protection.

Equine deep stromal abscesses (51 cases - 2004-2009)--Part 2: the histopathology and immunohistochemical aspect with attention to the histopathologic diagnosis, vascular response, and infectious agents.

PURPOSE To investigate histopathologic and immunohistochemical aspects of equine deep stromal abscesses (DSA) with a focus on the histopathologic diagnosis, presumptive etiology, and the immunohistochemical expression of three angiogenesis-related factors: vascular endothelial growth factor-A (VEGF-A), pigment epithelium-derived factor (PEDF), and interleukin-1 receptor antagonist (IL-1ra). SAMPLE POPULATION Paraffin-embedded biopsy samples from 51 DSA. The biopsies were collected from full-thickness penetrating keratoplasty or split-thickness lamellar keratoplasty surgeries at the University of Florida Veterinary Medical Center in the period from 2004 to 2009. PROCEDURE The histopathologic and immunohistochemical findings were tested for association between each other. Prevalence calculation and test for association with qualitative data analysis was used for data evaluation. RESULTS Fungal hyphae were found histologically in 47.1% (n = 24) of the DSA cases. Histopathologically, most fungal DSA showed suppurative keratitis (n = 34; 66.7%) and little to no stromal vascularization infiltrating the abscess (negative association, P = 0.005). All three angiogenesis-related factors were expressed to some degree in DSA tissue. A negative association between VEGF-A and PEDF when compared to the presence of fungal hyphae (P < 0.001, P = 0.023) indicated that cases positive for these two factors will most probably not have fungal hyphae present. CONCLUSION Abnormally decreased VEGF-A expression is suggested as the reason for the slow vascularization and delayed resolution of fungal DSA, whereas PEDF and IL-ra did not seem to have any influence on the vascularization process. Clinical and histopathologic characteristics of DSA make it possible to suggest an etiology for an equine DSA with an unknown etiology.

Utilization of Feline ELISPOT for Mapping Vaccine Epitopes

A commercial feline immunodeficiency virus (FIV) vaccine consisting of inactivated dual-subtype viruses was released in the USA in 2002 and released subsequently over the next 6 years in Canada, Australia, New Zealand, and Japan. Based on the genetic, morphologic, and biochemical similarities between FIV and human immunodeficiency virus-1 (HIV-1), FIV infection of domestic cats is being used as a small animal model of HIV/AIDS vaccine. Studies on prototype and commercial FIV vaccines provide new insights to the types of immunity and the vaccine epitopes required for an effective human HIV-1 vaccine. ELISPOT assays to detect cytokines, chemokines, and cytolytic mediators are widely used to measure the magnitude and the types of cellular immunity produced by vaccination. Moreover, such approach has identified regions on both HIV-1 and FIV proteins that induce robust antiviral cellular immunity in infected hosts. Using the same strategy, cats immunized with prototype and commercial FIV vaccines are being analyzed by feline interferon-γ and IL-2 ELISPOT systems to identify the vaccine epitope repertoire for prophylaxis.

Evolutionarily conserved T-cell epitopes on FIV for designing an HIV/AIDS vaccine.

This review will discuss the current state of the human HIV-1 vaccine trials including the safety consideration of vaccine composition and difficulties in determining and defining protective immunity and epitopes to HIV-1. Vaccines in animal models of lentivirus infection are compared. In particular, the findings from the prototype FIV vaccine and the HIV-1 protein immunizations studies in cats are discussed, as well as the resulting research regarding a potential HIV-1 vaccine design based on evolutionarily conserved T-cell epitopes.

An initial examination of the potential role of T-cell immunity in protection against feline immunodeficiency virus (FIV) infection

The importance of vaccine-induced T-cell immunity in conferring protection with prototype and commercial FIV vaccines is still unclear. Current studies performed adoptive transfer of T cells from prototype FIV-vaccinated cats to partial-to-complete feline leukocyte antigen (FLA)-matched cats a day before either homologous FIVPet or heterologous-subtype pathogenic FIVFC1 challenge. Adoptive-transfer (A-T) conferred a protection rate of 87% (13 of 15, p < 0.001) against FIVPet using the FLA-matched T cells, whereas all 12 control cats were unprotected. Furthermore, A-T conferred protection rate of 50% (6 of 12, p<0.023) against FIVFC1 using FLA-matched T cells, whereas all 8 control cats were unprotected. Transfer of FLA-matched T and B cells demonstrated that T cells are needed to confer A-T protection. In addition, complete FLA-matching and addition of T-cell numbers > 13 × 10(6) cells were required for A-T protection against FIVFC1 strain, reported to be a highly pathogenic virus resistant to vaccine-induced neutralizing-antibodies. The addition of FLA-matched B cells alone was not protective. The poor quality of the anti-FIV T-cell immunity induced by the vaccine likely contributed to the lack of protection in an FLA-matched recipient against FIVFC1. The quality of the immune response was determined by the presence of high mRNA levels of cytolysin (perforin) and cytotoxins (granzymes A, B, and H) and T helper-1 cytokines (interferon-γ [IFNγ] and IL2). Increased cytokine, cytolysin and cytotoxin production was detected in the donors which conferred protection in A-T studies. In addition, the CD4(+) and CD8(+) T-cell proliferation and/or IFNγ responses to FIV p24 and reverse transcriptase increased with each year in cats receiving 1X-3X vaccine boosts over 4 years. These studies demonstrate that anti-FIV T-cell immunity induced by vaccination with a dual-subtype FIV vaccine is essential for prophylactic protection against AIDS lentiviruses such as FIV and potentially HIV-1.

Development and validation of a novel hydrolysis probe real-time polymerase chain reaction for agamid adenovirus 1 in the central bearded dragon (Pogona vitticeps):

Agamid adenovirus 1 (AgAdv-1) is a significant cause of disease in bearded dragons (Pogona sp.). Clinical manifestations of AgAdv-1 infection are variable and often nonspecific; the manifestations range from lethargy, weight loss, and inappetence, to severe enteritis, hepatitis, and sudden death. Currently, diagnosis of AgAdv-1 infection is achieved through a single published method: standard nested polymerase chain reaction (nPCR) and sequencing. Standard nPCR with sequencing provides reliable sensitivity, specificity, and validation of PCR products. However, this process is comparatively expensive, laborious, and slow. Probe hybridization, as used in a TaqMan assay, represents the best option for validating PCR products aside from the time-consuming process of sequencing. This study developed a real-time PCR (qPCR) assay using a TaqMan probe–based assay, targeting a highly conserved region of the AgAdv-1 genome. Standard curves were generated, detection results were compared with the gold standard conventional PCR and sequencing assay, and limits of detection were determined. Additionally, the qPCR assay was run on samples known to be positive for AgAdv-1 and samples known to be positive for other adenoviruses. Based on the results of these evaluations, this assay allows for a less expensive, rapid, quantitative detection of AgAdv-1 in bearded dragons.

Pegylated feline granulocyte colony-stimulating factor increases neutrophil levels in cats.

Neutropenia can often be corrected by treatment with granulocyte-colony stimulating factor (G-CSF) and off-label use of commercial human G-CSF (HuG-CSF) is a commonly used treatment for neutropenic animals. However, long-term HuG-CSF treatment can be associated with adverse effects, including neutropenia. Here, feline (Fe) G-CSF was produced in Pichia pastoris, pegylated (Peg) FeG-CSF and tested in cats. A randomized controlled clinical trial was conducted to evaluate the efficacy of PegFeG-CSF compared to FeG-CSF or HuG-CSF in FIV-infected (n=14), FIV-uninfected healthy cats (n=19), and in HuG-CSF-induced neutropenic cats (n=4). Daily FeG-CSF doses induced higher neutrophil production than HuG-CSF after the second week of treatment (P ⩽ 0.002). Weekly doses of PegFeG-CSF induced higher neutrophil counts and showed greater sustained activity than weekly doses of FeG-CSF. PegFeG-CSF provided the most therapeutic and sustainable neutrophil production (P<0.001) in both FIV-uninfected and FIV-infected cats, without the development of neutralizing antibodies. Conversely, all HuG-CSF-treated cats developed neutralizing antibodies, suggesting cross-reactive antibodies to endogenous G-CSF in a majority of the cases with severe neutropenia. Strikingly, when PegFeG-CSF was used to rescue cats with HuG-CSF-induced neutropenia, clinically normal neutrophil numbers returned. Thus, PegFeG-CSF appears to be a superior treatment for neutropenia in feline patients.

Fast Detection of Perkinsus Marinus, a Prevalent Pathogen of Oysters and Clams from Sea Waters

Antibody-functionalized, Au-gated AlGaN/GaN high electron mobility transistors (HEMTs) were used to detect Perkinsus marinus. The antibody was anchored to the gate area through immobilized thioglycolic acid. The AlGaN/GaN HEMT were grown by a molecular beam epitaxy system (MBE) on sapphire substrates. Infected sea waters were taken from the tanks in which Tridacna crocea infected with P. marinus were living and dead. The AlGaN/GaN HEMT showed a rapid response of drain-source current in less than 5 seconds when the infected sea waters were added to the antibody-immobilized surface. The recyclability of the sensors with wash buffers between measurements was also explored. These results clearly demonstrate the promise of field-deployable electronic biological sensors based on AlGaN/GaN HEMTs for Perkinsus marinus detection.