Immunization with a vaccine combining herpes simplex virus 2 (HSV-2) glycoprotein C (gC) and gD subunits improves the protection of dorsal root ganglia in mice and reduces the frequency of recurrent vaginal shedding of HSV-2 DNA in guinea pigs compared to immunization with gD alone

Attempts to develop a vaccine to prevent genital herpes simplex virus 2 (HSV-2) disease have been only marginally successful, suggesting that novel strategies are needed. Immunization with HSV-2 glycoprotein C (gC-2) and gD-2 was evaluated in mice and guinea pigs to determine whether adding gC-2 to a gD-2 subunit vaccine would improve protection by producing antibodies that block gC-2 immune evasion from complement. Antibodies produced by gC-2 immunization blocked the interaction between gC-2 and complement C3b, and passive transfer of gC-2 antibody protected complement-intact mice but not C3 knockout mice against HSV-2 challenge, indicating that gC-2 antibody is effective, at least in part, because it prevents HSV-2 evasion from complement. Immunization with gC-2 also produced neutralizing antibodies that were active in the absence of complement; however, the neutralizing titers were higher when complement was present, with the highest titers in animals immunized with both antigens. Animals immunized with the gC-2-plus-gD-2 combination had robust CD4+ T-cell responses to each immunogen. Multiple disease parameters were evaluated in mice and guinea pigs immunized with gC-2 alone, gD-2 alone, or both antigens. In general, gD-2 outperformed gC-2; however, the gC-2-plus-gD-2 combination outperformed gD-2 alone, particularly in protecting dorsal root ganglia in mice and reducing recurrent vaginal shedding of HSV-2 DNA in guinea pigs. Therefore, the gC-2 subunit antigen enhances a gD-2 subunit vaccine by stimulating a CD4+ T-cell response, by producing neutralizing antibodies that are effective in the absence and presence of complement, and by blocking immune evasion domains that inhibit complement activation.     

Mapping of B cell epitopes on the zona pellucida 2 protein of a marsupial, the brushtail possum (Trichosurus vulpecula)

Immunocontraceptive vaccines against zona pellucida (ZP) proteins are being developed for brushtail possum (Trichosurus vulpecula) management in New Zealand. Mapping of B cell epitopes on the ZP2 protein of possums was undertaken in this study to define the antigenic regions that may be crucial to sperm-egg binding. The amino acid sequence of the full-length possum ZP2 protein (712 amino acids) was used to synthesize a complete set of 71 (15-mer) biotinylated peptides with an offset of five amino acids. The peptides were used in a modified enzyme-linked immunosorbent assay (ELISA) to identify continuous epitopes recognized by antibodies in the sera of possums immunized with recombinant possum ZP2 (rZP2) constructs. Seventeen continuous epitopes were located on possum ZP2 protein. Comparisons of the peptide binding pattern of antibodies in individual sera with the fertility status of the same immunized possums revealed three significant infertility-relevant peptide epitopes (amino acids 111-125, 301-315, and 431-445). One of these (amino acids 431-445) bound to possum spermatozoa from the caudal epididymis. The implications of these findings for developing immunocontraceptive vaccines for possum control are discussed.     

Host Range Restriction of Epstein-Barr Virus and Related Lymphocryptoviruses

Epstein-Barr-related herpesviruses, or lymphocryptoviruses (LCV), naturally infect humans and nonhuman primates (NHP), but their host range is not well characterized. Using LCV and B cells from multiple species of Hominidae and Cercopithecidae, we show that LCV can immortalize B cells from some nonnative species but that growth transformation is restricted to B cells from their own family of hominoids or Old World NHP, suggesting a high degree of LCV adaptation to their natural primate host.     

Phylogenetic Analysis of ADP-Glucose Pyrophosphorylase Subunits Reveals a Role of Subunit Interfaces in the Allosteric Properties of the Enzyme

ADP-glucose pyrophosphorylase (AGPase) catalyzes a rate-limiting step in glycogen and starch synthesis in bacteria and plants, respectively. Plant AGPase consists of two large and two small subunits that were derived by gene duplication. AGPase large subunits have functionally diverged, leading to different kinetic and allosteric properties. Amino acid changes that could account for these differences were identified previously by evolutionary analysis. In this study, these large subunit residues were mapped onto a modeled structure of the maize (Zea mays) endosperm enzyme. Surprisingly, of 29 amino acids identified via evolutionary considerations, 17 were located at subunit interfaces. Fourteen of the 29 amino acids were mutagenized in the maize endosperm large subunit (SHRUNKEN-2 [SH2]), and resulting variants were expressed in Escherichia coli with the maize endosperm small subunit (BT2). Comparisons of the amount of glycogen produced in E. coli, and the kinetic and allosteric properties of the variants with wild-type SH2/BT2, indicate that 11 variants differ from the wild type in enzyme properties or in vivo glycogen level. More interestingly, six of nine residues located at subunit interfaces exhibit altered allosteric properties. These results indicate that the interfaces between the large and small subunits are important for the allosteric properties of AGPase, and changes at these interfaces contribute to AGPase functional specialization. Our results also demonstrate that evolutionary analysis can greatly facilitate enzyme structure-function analyses.ADP-glucose pyrophosphorylase (AGPase) catalyzes the conversion of Glc-1-P (G-1-P) and ATP to ADP-Glc and pyrophosphate. This reaction represents a rate-limiting step in starch synthesis (Hannah, 2005). AGPase is an allosteric enzyme whose activity is regulated by small effector molecules. In plants, AGPase is activated by 3-phosphoglyceraldehyde (3-PGA) and deactivated by inorganic phosphate (Pi).Plant AGPase is a heterotetramer consisting of two identical large and two identical small subunits. The large and small subunits of AGPase were generated by a gene duplication. Subsequent sequence divergence has given rise to complementary rather than interchangeable subunits. Indeed, both subunits are needed for AGPase activity (Hannah and Nelson, 1976, Burger et al., 2003). Biochemical studies have indicated that both subunits are important for catalytic and allosteric properties (Hannah and Nelson, 1976; Greene et al., 1996a, 1996b; Ballicora et al., 1998; Laughlin et al., 1998; Frueauf et al., 2001; Kavakli et al., 2001a, 2001b; Cross et al., 2004, 2005; Hwang et al., 2005, 2006, 2007; Kim et al., 2007; Ventriglia et al., 2008). Surprisingly, Georgelis et al. (2007, 2008) showed that, in angiosperms, the small subunit is under greater evolutionary pressure compared with the large subunit. Detailed analyses have shown that the greater constraint on the small subunit is due to its broader tissue expression patterns compared with the large subunit and the fact that the small subunit must interact with multiple large subunits.Large subunits have undergone more duplication events than have small subunits (Georgelis et al., 2008). This has led to the creation of five groups of large subunits that differ in their patterns of tissue of expression (Akihiro et al., 2005; Crevillen et al., 2005; Ohdan et al., 2005). Crevillen et al. (2003) studied the biochemical properties of four Arabidopsis (Arabidopsis thaliana) AGPases consisting of the four different large subunits and the only functional small subunit in Arabidopsis. The different AGPases had different kinetic and allosteric properties. More specifically, the AGPases differed in their affinity for the allosteric regulator 3-PGA and the substrates G-1-P and ATP. This possibly reflects the different 3-PGA, G-1-P, and ATP levels in the various tissues. This evidence indicates that not only did the different large subunit groups subfunctionalize in terms of expression, but also these groups may have specialized in terms of protein function. While the study of Crevillen et al. (2003) pointed to functional specialization of the large subunit, the identity of the amino acid sites in the large subunit that account for these kinetic and allosteric differences was not pursued.Georgelis et al. (2008) presented supporting evidence for AGPase large subunit specialization by identifying positively selected amino acid sites in the phylogenetic branches following gene duplication events. We also identified amino acid residues that were conserved in one large subunit group but not conserved in another large subunit group (type I functional divergence; Gu, 1999) and amino acid residues that are conserved within large subunit groups but are variable among large subunit groups (type II functional divergence; Gu, 2006). Positively selected type I and type II sites could have contributed to specialization of the different large subunit groups. Indeed, positively selected type II sites in several proteins have been proven via site-directed mutagenesis (Bishop, 2005; Norrgård et al., 2006; Cavatorta et al., 2008; Courville et al., 2008) to be important for protein function and functional specialization. Additionally, several positively selected type I and type II amino acid sites in the large AGPase subunit identified in our previous evolutionary analysis (Georgelis et al., 2008) have been implicated in the kinetic and allosteric properties and heat stability of AGPase. The role of these sites was demonstrated by site-directed mutagenesis experiments of large subunits from Arabidopsis, maize endosperm, and potato (Solanum tuberosum) tuber (Ballicora et al., 1998, 2005; Kavakli et al., 2001a; Jin et al., 2005; Linebarger et al., 2005; Ventriglia et al., 2008). These analyses indicate that the rest of the amino acid sites identified as positive type I and type II sites in our previous evolutionary analysis (Georgelis et al., 2008) represent promising candidate targets for mutagenesis.To identify large subunit amino acids that are possibly important in controlling enzyme properties and that may have contributed to large subunit specialization, we conducted site-directed mutagenesis of the maize endosperm large subunit encoded by Shrunken-2 (Sh2). We specifically identified amino acids of SH2 that correspond to amino acid sites that were detected as positive type I and type II sites during the large subunit evolution (Georgelis et al., 2008). We then replaced the SH2 residues with amino acids of a group different from the SH2 family. Several amino acid sites important for the kinetic and allosteric properties and heat stability of AGPase were identified. Our results indicate that the subunit interfaces between the large and small subunits are important for the allosteric properties of AGPase. They also indicate that amino acid changes at subunit interfaces have been important for AGPase specialization in terms of allosteric properties. These experiments also support the idea that the majority of positively selected sites as detected by codon substitution models (Nielsen and Yang, 1998; Yang et al., 2000) and type II sites are not false positives. Site-directed mutagenesis of such sites can greatly facilitate enzyme structure-function analyses.     

FcR interactions do not play a major role in inhibition of experimental autoimmune encephalomyelitis by anti-CD154 monoclonal antibodies

It has been demonstrated that anti-CD154 mAb treatment effectively inhibits the development of experimental autoimmune encephalomyelitis (EAE). However, although it appears to prevent the induction of Th1 cells and reactivation of encephalitogenic T cells within the CNS, little information is available regarding the involvement of alternative mechanisms, nor has the contribution of Fc effector mechanisms in this context been addressed. By contrast, efficacy of anti-CD154 mAbs in models of allotransplantation has been reported to involve long-term unresponsiveness, potentially via activation of T regulatory cells, and recently was reported to depend on Fc-dependent functions, such as activated T cell depletion through FcgammaR or complement. In this study we demonstrate that anti-CD154 mAb treatment inhibits EAE development in SJL mice without apparent long-term unresponsiveness or active suppression of disease. To address whether the mechanism of inhibition of EAE by anti-CD154 mAb depends on its Fc effector interactions, we compared an anti-CD154 mAb with its aglycosyl counterpart with severely impaired FcgammaR binding and reduced complement binding activity with regard to their ability to inhibit clinical signs of EAE and report that both forms of the Ab are similarly protective. This observation was largely confirmed by the extent of leukocyte infiltration of the CNS; however, mice treated with the aglycosyl form may display slightly more proteolipid protein 139-151-specific immune reactivity. It is concluded that FcR interactions do not play a major role in the protective effect of anti-CD154 mAb in the context of EAE, though they may contribute to the full abrogation of peripheral peptide-specific lymphocyte responses.     

The potato tuber,maize endosperm and a chimeric maize-potato ADP-glucose pyrophosphorylase exhibit fundamental differences in Pi inhibition

ADP-glucose pyrophosphorylase (AGPase) is highly regulated by allosteric effectors acting both positively and negatively. Enzymes from various sources differ, however, in the mechanism of allosteric regulation. Here, we determined how the effector, inorganic phosphate (Pi), functions in the presence and absence of saturating amounts of the activator, 3-phosphoglyceric acid (3-PGA). This regulation was examined in the maize endosperm enzyme, the oxidized and reduced forms of the potato tuber enzyme as well as a small subunit chimeric AGPase (MP), which contains both maize endosperm and potato tuber sequences paired with a wild-type maize large subunit. These data, combined with our previous kinetic studies of these enzymes led to a model of Pi inhibition for the various enzymes. The Pi inhibition data suggest that while the maize enzyme contains a single effector site that binds both 3-PGA and Pi, the other enzymes exhibit more complex behavior and most likely have at least two separate interacting binding sites for Pi. The possible physiological implications of the differences in Pi inhibition distinguishing the maize endosperm and potato tuber AGPases are discussed.     

Chinook and Coho salmon hybrids linked to habitat and climatic changes on Vancouver Island,British Columbia

Between 2013 and 2019, 63 presumed Chinook salmon Oncorhynchus tshawytscha sampled primarily in the Strait of Georgia (0.63% of total sample) were identified as potential Chinook–Coho (Oncorhynchus kisutch) hybrids by the presence of anomalous microsatellite genotypes. Their hybrid origin was confirmed by single nucleotide polymorphism amplification of two species‐specific amplicons. Mitochondrial DNA indicated that most of these fish resulted from the hybridization of Coho salmon females and Chinook salmon males. Although no diagnostic external features were identified, several individuals displayed an abnormal scale arrangement on the caudal peduncle. One hybrid juvenile examined for meristics exhibited a pyloric caeca count intermediate between published values for Chinook and Coho salmon. Most hybrids originated in the Cowichan River during the 2014 brood year. Their prevalence in the watershed is a naturally occurring event, likely exacerbated by prolonged low water levels which limit habitat and delay Chinook salmon spawning, in addition to the differential abundance of the parental species. This research is the first to document ongoing natural hybridization (Chinook–Coho salmon crosses) and link it to habitat and climatic changes, and includes the identification of eight F1 adults and two juvenile backcross or F2 hybrids. The potential negative impacts of hybridization, particularly in Coho salmon through potential introgression, warrant hybrid identification as an ecosystem monitoring tool within a survey program.     

Application of the IUCN Red Listing system to setting species targets for conservation planning purposes

Biodiversity targets, or estimates of the quantities of biodiversity features that should be conserved in a region, are fundamental to systematic conservation planning. We propose that targets for species should be based on the quantitative thresholds developed for the Vulnerable category of the IUCN Red List system, thereby avoiding future listings of species in an IUCN Red List threat category or an increase in the extinction risk, or ultimate extinction, of species already listed as threatened. Examples of this approach are presented for case studies from South Africa, including threatened taxa listed under the IUCN Red List criteria of A to D, a species listed as Near Threatened, a species of conservation concern due to its rarity, and one species in need of recovery. The method gives rise to multiple representation targets, an improvement on the often used single representation targets that are inadequate for long term maintenance of biodiversity or the arbitrary multiple representation and percentage targets that are sometimes adopted. Through the implementation of the resulting conservation plan, these targets will ensure that the conservation status of threatened species do not worsen over time by qualifying for higher categories of threat and may actually improve their conservation status by eliminating the threat of habitat loss and stabilizing population declines. The positive attributes ascribed to the IUCN Red List system, and therefore to the species targets arising from this approach, are important when justifying decisions that limit land uses known to be detrimental to biodiversity.     

Disentangling the effects of global climate and regional land-use change on the current and future distribution of mangroves in South Africa

The mangrove distribution in South Africa is fragmented and restricted to small forest patches occupying only 16 % of the estuaries within the current range. In this study we used species distribution models to test (1) whether the absence of mangrove forest and its species (Avicennia marina, Bruguiera gymnorrhiza and Rhizophora mucronata) within their current range is driven by climate or by climate combined with human or geomorphic perturbation and (2) how climate change may potentially affect the latitudinal limit of the mangrove forests and its species in South Africa. We used three modelling techniques (generalized linear models, generalized additive models and gradient boosting machines) and a set of three climate-based predictive variables (minimum air temperature of the coldest month, waterbalance and growing-degree days) combined separately with an index of human or geomorphic perturbation. Climate variables for the future projections were derived from two general circulation models driven by two socio-economic scenarios (A2a and B2a). Within the range of the mangrove forest, the fragmented distribution of the mangroves in South Africa was not explained by our set of climate variables alone. The index of human perturbations slightly improved the predictions but the index of geomorphic perturbation did not. Climate change will create climatically suitable sites for the mangrove forest and the two species A. marina and B. gymnorrhiza beyond their current limits, but model outcomes did not agree on the future potential distribution of R. mucronata. We were able to successfully predict range limits and to detect future climatically suitable sites beyond the current limits. Factors controlling mangrove distribution within its range are still to be identified although absences were partly explained by human perturbations.