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The properties of human immunodeficiency virus type 1 (HIV-1) and its simian counterpart SIV that enable persistent replication in the face of robust cellular, antibody, and innate immune responses have complicated efforts to develop a safe and effective vaccine. Vaccine protection against HIV-1 infection may require a combination of immune mechanisms. However, the types of immune responses that can be induced by vaccination to prevent HIV-1 infection remain unclear. The features of the viral envelope glycoprotein (Env) that confer inherent resistance to neutralization by antibodies also interfere with the development of antibody responses. We therefore vaccinated rhesus macaques with single-cycle SIV (scSIV) strains expressing Env proteins mutated to remove features that interfere with the induction of antibody responses. Antibodies capable of neutralizing Env-modified but not wild-type SIV were selectively enhanced. Identifying the immune responses underlying complete protection by live-attenuated SIV against pathogenic SIV challenge may provide guidance for HIV-1 vaccine design. To test the hypothesis that antibodies not measurable by assays for virus neutralization correlate with protection by live-attenuated SIV, we developed a novel assay for antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC activity increased progressively over time after inoculation, and was measurable against viruses expressing heterologous Env proteins from independent SIV isolates when neutralization was undetectable. Two separate pathogenic \(SIV_{mac}251\) challenge experiments took advantage of either the strain specificity or the time-dependent development of immunity to overcome complete protection by live-attenuated SIV. In both experiments, macaques inoculated with live-attenuated SIV that remained uninfected by \(SIV_{mac}251\) had significantly higher ADCC activity than those that became infected. We also measured ADCC for the primary immune correlates analysis of a recent HIV-1 vaccine clinical trial in Thailand (RV144) that reported modest vaccine protection (31%). There was a nonsignificant trend towards lower risk of infection among vaccinees with high versus low relative ADCC activity. However, Env-specific IgA correlated with risk, prompting an analysis stratified by IgA levels. Among vaccinees with low Env-specific IgA, there was lower risk of infection among those with higher ADCC activity. These observations suggest that antibodies that direct ADCC may contribute to vaccine protection against SIV and HIV-1 infection.

Edwardsiella ictaluri causes enteric septicemia of catfish (ESC), one of the most important bacterial diseases of farmed channel catfish in the USA. Use of live attenuated vaccines (LAVs) is an effective strategy for combating mortalities in catfish farms. Our research group has developed three live attenuated E. ictaluri strains [EiΔevpB, EiΔgcvPΔsdhCΔfrdA (ESC-NDKL1), and EiΔhemRΔfrdAΔsdhC (triple-hemR)] that provide various levels of protection against ESC. However, the protective mechanisms of these vaccine candidates are mostly unknown. The overall objective of my study was to investigate protective mechanisms of these LAVs. To accomplish this, catfish fry were immersion challenged with EiΔevpB, ESC-NDKL1, and triple-hemR. Additional catfish fry were immersion challenged with Aquavac-ESC and E. ictaluri wild-type (EiWT) as controls. The internalization of antigens through the mucosal surfaces as well as the pathology and molecular immune responses were studied. The investigations showed that EiΔevpB and ESC-NDKL1 were highly safe and efficacious compared to Aquavac-ESC and EiWT. Pathologically, vaccination with EiΔevpB and ESC-NDKL1 decreased the pathological lesions, EiWT replication in catfish, and increased the ability of the immune system to resist and kill EiWT. On the other hand, triple-hemR was not safe causing severe tissue damage similar to EiWT. The gene expression data showed significantly high expression of innate and adaptive immune genes following vaccination and challenge with EiWT. Additionally, EiΔevpB and ESC-NDKL1 induced immune proliferation in pronephros after exposure to EiWT. Taken together, EiΔevpB and ESC-NDKL1 vaccine strains performed better to induce immune responses and reduce damage in the host tissues during EiWT challenge compared to Aquavac-ESC.

Virulent Aeromonas hydrophila (vAh) is a causative agent of motile Aeromonas septicemia (MAS) in catfish. There are limitations in the current therapeutic and preventative strategies against vAh. The pathogenesis of MAS as well as the immune response of catfish to vAh infection are poorly understood. The aim of this study is to: 1) develop a dual live attenuated vaccine against MAS and enteric septicemia of catfish caused by Edwardsiella ictaluri; and 2) evaluate the vAh bacterial load and gene expression patterns in catfish tissues following vAh infection. Previously, six recombinant vAh proteins (outer membrane protein, TonB-dependent receptor, three fimbrial proteins, and an ATPase) were identified to have vaccine efficacy against MAS, and live attenuated E. ictaluri vaccine strain ESC-NDKL1 was identified as an effective vector for expressing vAh antigens. A total of 29 recombinant ESC-NDKL1 strains have been constructed with the integration of one, two, or three genes encoding vAh antigens into the ESC-NDKL1 chromosome. Vaccine efficacy of the constructed strains was evaluated in channel catfish fingerlings. Four recombinant ESC-NDKL1 strains expressing two vAh antigens (ESC-NDKL1::atpase::fimMrfG, ESC-NDKL1::fim::fimMrfG, ESC-NDKL1::tdr::fimMrfG, and ESC-NDKL1::fim::ompA) showed significant protection against MAS with relative percent of survival (RPS) values of 55.72%, 60.18%, 61.74%, and 54.81%. Four triple recombinant ESC-NDKL1 strains (ESC-NDKL1::fimMrfG::ompA::fimA, ESC-NDKL1::atpase::fimMrfG::ompA, ESC-NDKL1::fim::fimMrfG::ompA and ESC-NDKL1::atpase::tdr::fim) showed the best protection with RPS values of 77.93%, 63.18%, 67.74%, and 82.35%. To gain a better understanding of vAh pathogenesis, catfish fingerlings were intraperitoneally injected with vAh strain ML09-119. The anterior kidney, liver, and spleen were collected for determination of vAh distribution and expression of thirteen pro-inflammatory, innate, and adaptive immune-related genes using real-time PCR. Results revealed that vAh spread into catfish tissues within 2 hours and peaked at 12 hours post-infection. vAh infection initiated a strong inflammatory response in catfish tissues. Additionally, our research revealed that surviving catfish were able to develop a primary immune response and possibly generation of memory B cells against MAS. Such information will facilitate the development of vaccines and therapeutic drugs for preventing and treating MAS outbreaks in catfish aquaculture.

Catfish aquaculture is the largest cultured food fish industry in the United States, accounting for approximately $375 million in sales annually, with Mississippi leading the industry with 36,200 surface acres of production. However, infectious diseases such as enteric septicemia of catfish (ESC) are decreasing production efficiencies, creating losses of $40-60 million annually. Live-attenuated oral ESC vaccines are effective in preventing ESC infections, but have not been widely adopted by the catfish industry due to the lack of a system to administer the oral vaccine at the scale seen in commercial catfish production systems. An automated system was developed to administer a dosage of 220.5 ml of a live-attenuated ESC vaccine per kg of catfish feed, adapting commercial catfish feeder design to include a screw conveyor for mixing vaccine and feed in a continuous process, pulse-width modulated spray nozzle control for precise vaccine application, and a programmable automation controller to regulate and monitor system performance. Initial performance evaluations demonstrated system operation within the desired design specifications. System feed rates were determined to be a function of the rotational speed (RPM) of the screw conveyor and to be linear across the operational range. Feed rates were observed to decrease by 45% over dry feed when applying liquid vaccine to the feed stream at the 220.5 ml/kg (100 ml/lb) rate, resulting in a feed rate range of 6.80-34.02 kg/min (15-75 lb/min) (95% CI). Uniform pellet-level vaccine distribution is crucial to efficacy as pellet consumption is directly correlated with fish size, with more criticality in smaller fish fed at low rates. Pellet vaccine concentrations at 6.80, 20.41, and 34.02 ml/kg were highly variable and vaccine application at all rates were observed to be statistically different (less) than the target 220.5ml/kg rate (95% CI), pointing to potential issues with vaccine delivery system configuration or inadequacies in sampling methodology. Further evaluation at the pellet level with live-attenuated vaccine to obtain viable cell counts within individual pellets would provide data necessary to address uniformity of coverage questions more fully and to develop operational protocols that maximize system capabilities and vaccine efficacy.

Vaccination against intracellular pathogens requires generation of pool of memory T cells, which can respond upon infection and mediate immune responses by either killing of infected host cells or induce killing mechanisms in infected cells. T cell-inducing vaccines aim to deliver the antigen to antigen presenting cells (APCs) by presenting on MHC molecules thus bridging innate and adaptive immunity. The intracellular pathogen Edwardsiella ictaluri causes enteric septicemia of catfish (ESC), which is a devastating disease in catfish industry. E. ictaluri can survive in professional phagocytes and use them as an infection source. Two new live attenuated vaccine (LAV) strains, EiDELTAevpB and ESC-NDKL, were developed by our group. However, the role of LAVs in phagocytosis, bacterial killing, and antigen presentation is unexplored. Therefore, further research is necessary to determine immune responses in channel catfish against LAVs. The long-term goal of this project is to identify immunological APC-dependent mechanisms that underscore E. ictaluri pathogenesis to enable development of effective control strategies for ESC. The overall goal of this project is to assess the role of three professional APCs, dendritic cells (DCs), macrophages and B cells in the LAV-induced innate and adaptive immune responses in catfish. The central hypothesis is that efficacious LAV strains will enhance phagocytosis and microbial killing, and promote the generation of T cells that regulate and control protective B cell-mediated immunity. The rationale for this research is that more detailed knowledge about phenotype and function of catfish APCs will not only help gain insight into the evolution of vertebrate adaptive immune system but will provide valuable information for development and optimization of immunotherapies and vaccination protocols for aquaculture use. In this study, we first identified DC-like cells in immune-related organs of catfish and assessed their expression patterns in lymphoid organs of catfish in E. ictaluri infection. Although WT strain induces the functional inability of DC-like cells in migration and maturation, LAVs strains promote the migration and maturation of DC-like cells for antigen presentation. Two LAVs enhanced the phagocytosis and killing activity in catfish macrophages and B cells. Also, LAVs induce high expression of T cell-related genes without causing inflammation.

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