a-technique-to-screen-american-beech-for-resistance-to-beech-scale

A Technique to Screen American Beech for Resistance to the Beech Scale Insect (<em>Cryptococcus fagisuga</em> Lind.)

Beech bark disease (BBD) results in high levels of initial mortality, leaving behind survivor trees that are greatly weakened and deformed.&#160;The ...

US Forest Service

We evaluated the efficacy of fenbendazole (FBZ) and milbemycin oxime (MO) in the treatment of baboons (Papio cynocephalus anubis) with naturally acquired Trichuris trichiura infection by comparing fecal egg count reduction (FECR) tests. We assigned 7 baboons, each singly housed and confirmed infected with T. trichiura, to treatment groups of FBZ (n=3) or MO (n=3), or as a control (n=1). All (100%) baboons that received FBZ stopped shedding T. trichiura eggs within 6 d of treatment, and fecal egg counts remained negative at 65 d after treatment. Although the number of T. trichiura eggs shed per gram of feces from 2 (67%) baboons decreased significantly after the second treatment with MO, this regimen never totally eliminated eggs of T. trichiura. The results of our study indicate that FBZ was more effective for treating baboons with T. trichiura than was MO.

Efficacy of fenbendazole and milbemycin oxime for treating baboons (Papio cynocephalus anubis) infected with Trichuris trichiura.

We designed an amebiasis subunit vaccine that is constructed by using four peptide epitopes of the galactose-inhibitable lectin heavy subunit that were recognized by intestinal secretory immunoglobulin A (IgA) antibodies from immune human subjects. These epitopes are contained in the region encompassing amino acids 758 to 1134 of the lectin heavy subunit, designated LC3. Baboons (Papio anubis) are natural hosts for Entamoeba histolytica; naturally infected baboons raised in captivity possess serum IgA antibodies to the same four LC3 epitopes as humans. Uninfected, seronegative baboons received four intranasal immunizations at 7-day intervals with the synthetic peptide vaccine (400, 800, or 1,600 mug per nostril) with cholera toxin (20 mug) as the adjuvant. As determined by an enzyme-linked immunosorbent assay (ELISA), each dose of the peptide vaccine elicited antipeptide serum IgA and IgG and intestinal IgA antibody responses in all six immunized baboons by day 28, 7 days after the last immunization (P, <0.01 for each dose compared to the cholera toxin control). The peptide vaccine elicited serum IgG and intestinal IgA antibodies that recognized purified recombinant LC3 protein (P, <0.008 and 0.02, respectively) and native lectin protein (P < 0.01). In addition, an indirect immunofluorescence assay with whole trophozoites (P < 0.01) and Western blot analysis confirmed that serum IgG antibodies from vaccinated baboons recognized native lectin protein on the surfaces of axenic E. histolytica trophozoites or from solubilized amebae. All four synthetic peptides were immunogenic; the vaccine elicited dose- and time-dependent responses, as determined by ELISA optical density readings indicating the production of serum and intestinal antibodies (P, <0.02 for antipeptide and antilectin antibodies). As a positive control, intranasal immunization with purified recombinant LC3 protein with cholera toxin as the adjuvant elicited a serum anti-LC3 IgA and IgG antibody response (P, 0.05 and <0.0001, respectively); however, no intestinal anti-LC3 IgA antibody response was observed (P = 0.4). Of interest, serum IgA and IgG antibodies elicited by the recombinant LC3 vaccine did not recognize any of the four putatively protective LC3 peptide epitopes. Both serum and fecal antibodies elicited by the peptide vaccine exhibited neutralizing activity, as determined by their dose-dependent inhibition of the galactose-specific adherence of E. histolytica trophozoites to Chinese hamster ovary cells in vitro (P, <0.001 for each group of antibodies compared to the control). In summary, a lectin-based intranasal polylysine-linked synthetic peptide vaccine was effective in eliciting an adherence-inhibitory, intestinal antilectin IgA antibody response in baboons. Future studies with the baboon model will determine vaccine efficacy against asymptomatic E. histolytica intestinal infection.

Adherence-inhibitory intestinal immunoglobulin a antibody response in baboons elicited by use of a synthetic intranasal lectin-based amebiasis subunit vaccine.

The peroxide response transcriptional regulator, PerR, is thought to contribute to virulence of group A Streptococcus (GAS); however, the specific mechanism through which it enhances adaptation for survival in the human host remains unknown. Here, we identify a critical role of PerR-regulated gene expression in GAS phagocytosis resistance and in virulence during pharyngeal infection. Deletion of perR in M-type 3 strain 003Sm was associated with reduced resistance to phagocytic killing in human blood and by murine macrophages in vitro. The increased phagocytic killing of the perR mutant was abrogated in the presence of the general oxidative burst inhibitor diphenyleneiodonium chloride (DPI), a result that suggests PerR-dependent gene expression counteracts the phagocyte oxidative burst. Moreover, an isogenic perR mutant was severely attenuated in a baboon model of GAS pharyngitis. In competitive infection experiments, the perR mutant was cleared from two animals at 24 h and from four of five animals by day 14, in sharp contrast to wild-type bacteria that persisted in the same five animals for 28 to 42 d. GAS genomic microarrays were used to compare wild-type and perR mutant transcriptomes in order to characterize the PerR regulon of GAS. These studies identified 42 PerR-dependent loci, the majority of which had not been previously recognized. Surprisingly, a large proportion of these loci are involved in sugar utilization and transport, in addition to oxidative stress adaptive responses and virulence. This finding suggests a novel role for PerR in mediating sugar uptake and utilization that, together with phagocytic killing resistance, may contribute to GAS fitness in the infected host. We conclude that PerR controls expression of a diverse regulon that enhances GAS resistance to phagocytic killing and allows adaptation for survival in the pharynx.

PerR confers phagocytic killing resistance and allows pharyngeal colonization by group A Streptococcus.

The prophylactic efficacy of a schistosome antigen (Sm-p80) was tested in a nonhuman primate model, the baboon. Using a total of 28 baboons, different vaccination strategies were used including recombinant Sm-p80 protein formulated in Toll-like receptor 7 and Toll-like receptor 9 agonists, and DNA priming followed by boosting with protein plus adjuvants. Recombinant protein approaches provided levels of prophylactic efficacy of 52%-58%, whereas prime-boost approaches conferred 38%-47% protection in baboons. An appropriately balanced pro-inflammatory (T-helper 17 [Th17] and Th1) and anti-inflammatory (Th2) type of response was generated; the Th1 and Th17 types of immune responses appear to be indicative of increased prophylactic efficacy. Production and expression of several cytokines (interleukin 2 [IL-2], interferon γ, IL-12α, IL-1β, IL-6, and IL-22) were up-regulated in vaccinated animals. Human correlate studies revealed Sm-p80 reactivity with immunoglobulin G in human serum samples from schistosome-infected individuals. In addition, a complete lack of prevailing Sm-p80-specific immunoglobulin E in a high-risk or infected population was observed, thus minimizing the risk of hypersensitivity reaction following vaccination with Sm-p80 in humans. This study provided the proof of concept to move Sm-p80 forward into further preclinical development leading to human clinical trials.

Preclinical prophylactic efficacy testing of Sm-p80-based vaccine in a nonhuman primate model of Schistosoma mansoni infection and immunoglobulin G and E responses to Sm-p80 in human serum samples from an area where schistosomiasis is endemic.

The ability of the Schistosoma mansoni antigen, Sm-p80, to provide cross-species protection against Schistosoma haematobium challenge was evaluated in hamster and baboon models. Pronounced reduction in worm burden (48%) and in tissue egg load (64%) was observed in hamsters vaccinated with recombinant Sm-p80 admixed with glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE). Similarly, in baboons, the Sm-p80/GLA-SE vaccine produced a 25% reduction in S. haematobium adult worms and decreased the egg load in the urinary bladder by 64%. A 40% and 53% reduction in fecal and urine egg output, respectively, was observed in vaccinated baboons. A balanced pro-inflammatory (Th17 and Th1) and Th2 type of response was generated after vaccination and appears indicative of augmented prophylactic efficacy. These data on cross-species protection coupled with the prophylactic, therapeutic and antifecundity efficacy against the homologous parasite, S. mansoni, reinforces Sm-p80 as a promising vaccine candidate. It is currently being prepared for GMP-compliant manufacture and for further pre-clinical development leading to human clinical trials. These results solidify the expectation that the Sm-p80 vaccine will provide relief for both the intestinal and the urinary schistosomiasis and thus will be greatly beneficial in reducing the overall burden of schistosomiasis.

Cross-species protection: Schistosoma mansoni Sm-p80 vaccine confers protection against Schistosoma haematobium in hamsters and baboons.

No vaccines are available for human use for any parasitic infections, including the helminthic disease schistosomiasis. Sm-p80, the large subunit of Schistosoma mansoni calpain, is a leading antigen candidate for a schistosomiasis vaccine. Prophylactic and antifecundity efficacies of Sm-p80 have been tested using a variety of vaccine approaches in both rodent and nonhuman primate models. However, the therapeutic efficacy of a Sm-p80-based vaccine had not been determined. In this study, we evaluated the therapeutic efficacy of Sm-p80 by using 2 different strategies and 3 Sm-p80-based vaccine formulations in baboons. Vaccine formulations were able to decrease established adult worms by 10%-36%, reduce retention of eggs in tissues by 10%-57%, and decrease egg excretion in feces by 13%-33%, compared with control formulations. Marked differences were observed in B and T cell immune correlates between vaccinated and control animals. This is the first report of killing of established adult schistosome worms by a vaccine. In addition to distinct prophylactic efficacy of Sm-p80, this study adds to the evidence that Sm-p80 is a potentially important antigen with both substantial prophylactic and therapeutic efficacies. These data reinforce that Sm-p80 should be moved forward along the path toward human clinical trials.

Use of an Sm-p80-Based Therapeutic Vaccine to Kill Established Adult Schistosome Parasites in Chronically Infected Baboons.

Non-human primates, such as baboons (Papio hamadryas anubis), are natural hosts for Entamoeba species; infections can be asymptomatic or result in invasive lethal disease. It was sought to determine whether following natural infection by Entamoeba. histolytica, baboon anti-amebic antibodies recognized native Gallectin, a recombinant portion of the lectin heavy subunit (designated LC3) and specific heavy subunit epitopes; we compared the specificity of anti-amebic antibodies from baboons to that of humans following asymptomatic E. histolytica infection or cure of amebic liver abscess (ALA). Female baboons (n=54), aged one to three years of age and living in captivity were screened for infection by real time PCR. E. histolytica infection was found in 37 baboons and was associated with serum anti-LC3 IgG (73%) and anti-LC3 IgA (46%) or intestinal anti-Gal-Lectin IgA antibody responses (49%), p<0.021 for each compared to that observed with baboons having an E. dispar infection (n=10) or uninfected baboons (n=7). The ELISA OD reading for anti-LC3 or anti-lectin antibodies correlated strongly with the presence of a PCR CT value indicative of E. histolytica infection. In humans with asymptomatic E. histolytica infection or those recently cured of ALA, 63% and 57% had serum anti- LC3 IgA and 65% and 57% had serum anti-LC3 IgG antibodies respectively. Epitope- specific synthetic peptides were used as capture antigens in ELISA; for baboons that possessed anti-LC3 and anti-lectin antibodies, 74% had anti-peptide IgG or IgA antibodies, compared to 86% of asymptomatic humans and 92% of ALA subjects(P>0.05).

Natural infection of baboons by Entamoeba histolytica elicits anti- gal-lectin heavy subunit IgA and IgG antibodies with shared epitope specificity to that of humans.

Based on data obtained using vaccine efficacy studies in mice, hamsters, and baboons, the credentials of Sm-p80 as a first tier vaccine candidate for schistosomiasis have been well established. Sm-p80-based vaccine formulation(s) have consistently exhibited potent prophylactic efficacy in reducing adult worm burden following cercarial challenge and induce killing of established adult worms in chronic infection. This vaccine is protective against both intestinal and urinary schistosomiasis. In this study, the longevity of Sm-p80-specific antibody responses was studied in mice and in baboons. Robust antibody titers were detected in mice for up to 60 weeks following vaccination with Sm-p80 recombinant vaccine (Sm-p80 + GLA-SE). In the follow-up experiments to our published studies, Sm-p80-specific IgG was also detected in baboons 5-8 years following the initial vaccination with an Sm-p80 DNA vaccine. In one baboon, transfer of Sm-p80-specific antibody was detected in umbilical cord blood and in the baby. These long-lasting humoral immune response data coupled with the vaccine efficacy data in rodents and nonhuman primates further strengthens the case for Sm-p80 to be moved forward through development leading to human clinical trials.

Longevity of Sm-p80-specific antibody responses following vaccination with Sm-p80 vaccine in mice and baboons and transplacental transfer of Sm-p80-specific antibodies in a baboon.

David W. Carey

Northern Research Station

US Forest Service

A Technique to Screen American Beech for Resistance to the Beech Scale Insect (Cryptococcus fagisuga Lind.)