Gregory J. Tobin

Crystal structure of plant aspartic proteinase prophytepsin: inactivation and vacuolar targeting

We determined at 2.3 Å resolution the crystal structure of prophytepsin, a zymogen of a barley vacuolar aspartic proteinase. In addition to the classical pepsin‐like bilobal main body of phytepsin, we also traced most of the propeptide, as well as an independent plant‐specific domain, never before described in structural terms. The structure revealed that, in addition to the propeptide, 13 N‐terminal residues of the mature phytepsin are essential for inactivation of the enzyme. Comparison of the plant‐specific domain with NK‐lysin indicates that these two saposin‐like structures are closely related, suggesting that all saposins and saposin‐like domains share a common topology. Structural analysis of prophytepsin led to the identification of a putative membrane receptor‐binding site involved in Golgi‐mediated transport to vacuoles.

Antibody repertoire development in fetal and neonatal piglets XXI. Usage of most VH genes remains constant during fetal and postnatal development

Usage of variable region gene segments during development of the antibody repertoire in mammals is unresolved in part because of the complexity of the locus in mice and humans and the difficulty of distinguishing intrinsic from extrinsic influences in these species. We present the first vertical studies on VH usage that spans the fetal and neonatal period using the piglet model. We tracked VH usage in DNA rearrangements and in VDJ transcripts throughout 75 days of gestation (DG) in outbred fetuses, thereafter in outbred germfree and colonized isolator piglets, isolator piglets infected with swine influenza and in conventionally reared nematode-infected adults. Seven VH genes account for >90% of the pre-immune repertoire which is the same among tissues and in both transcripts and DNA rearrangements. Statistical modeling supports the view that proportional usage of the major genes remains constant during fetal life and that postnatal usage ranking is similar to that during fetal life. Changes in usage ranking are developmental not antigen dependent. In this species exposure to environmental antigens results in diversification of the repertoire by somatic hypermutation of the same small number of VH genes that comprise the pre-immune repertoire, not by using other VH gene available in the germline. Therefore in swine a small number of VH genes shape the antibody repertoire throughout life questioning the need for extensive VH polygeny.

DNA immunization of mice and macaques with plasmids encoding hepatitis C virus envelope E2 protein expressed intracellularly and on the cell surface.

We analyzed the humoral immune response elicited by hepatitis C virus (HCV) E2 protein expressed in vivo after injection of plasmid DNA into mice and rhesus macaques. Three plasmids were used for immunization: a plasmid containing the entire sequence of the E2 and p7 genes (pE2); a plasmid encoding a truncated form of the E2 protein targeted to the cell surface (pE2surf); a control plasmid (pDisplay) lacking an HCV insert. Each plasmid was injected intramuscularly into 5 mice and intraepidermally (via gene gun) into 5 mice. Immunization was repeated three times at three week intervals. Five macaques were injected intramuscularly (two with pE2, two with pE2surf and one with pDisplay) and immunization was repeated after 8 weeks. All mice immunized via gene gun with pE2 or pE2surf developed anti-E2. The animals immunized with pE2surf developed an earlier and stronger humoral immune response than those immunized with pE2. Only 2 of the mice injected by the intramuscular route, both immunized with pE2surf, developed detectable anti-E2. One of the two macaques immunized with pE2 and both macaques immunized with pE2surf developed anti-E2; the humoral immune response was much stronger in the animals immunized with pE2surf. Our results suggest that presentation of HCV E2 on the cell surface may increase its immunogenicity while preserving its ability to react with antibodies generated during a natural infection.

Transport and Activation of the Vacuolar Aspartic Proteinase Phytepsin in Barley (Hordeum vulgare L.)

The primary translation product of barley aspartic proteinase, phytepsin (EC 3.4.23.40), consists of a signal sequence, a propart, and mature enzyme forms. Here, we describe post-translational processing and activation of phytepsin during its transport to the vacuole in roots, as detected by using metabolic labeling and immunoprecipitation. After removal of the signal sequence, the glycosylated precursor of 53 kDa (P53) was produced and further processed to polypeptides of 31 and 15 kDa (P31 + P15) and, subsequently, to polypeptides of 26 and 9 kDa (P26 + P9), 45 min and 24 h after synthesis, respectively. The processing occurred in a late-Golgi compartment or post-Golgi compartment, because brefeldin A inhibited the processing, and P53 acquired partial endoglycosidase H resistance 30 min after synthesis, whereas P15 was completely resistant. The N-glycosylation inhibitor tunicamycin had no effect on transport, but the absence of glycans on P53 accelerated the proteolytic processing. Phytepsin was also expressed in baculovirus-infected insect cells. The recombinant prophytepsin underwent autoproteolytic activation in vitro and showed enzymatic properties similar to the enzyme purified from grains. However, a comparison of the in vitro/in vivoprocessing sites revealed slight differences, indicating that additional proteases are needed for the completion of the maturationin vivo.

Antibody repertoire development in fetal and neonatal piglets. XXII. λ rearrangement precedes κ rearrangement during B‐cell lymphogenesis in swine

VDJ and VJ rearrangements, expression of RAG‐1, Tdt and VpreB, and the presence of signal joint circles (SJC) were used to identify sites of B‐cell lymphogenesis. VDJ, VλJλ but not VκJκ rearrangements or SJC were recovered from yolk sac (YS) at 20 days of gestation (DG) along with strong expression of VpreB and RAG‐1 but weak Tdt expression. VλJλ rearrangements but not VκJκ rearrangements were recovered from fetal liver at 30–50 DG. SJC were pronounced in bone marrow at 95 DG where VκJκ rearrangements were first recovered. The VλJλ rearrangements recovered at 20–50 DG used some of the same Vλ and Jλ segments seen in older fetuses and adult animals. Hence the textbook paradigm for the order of light‐chain rearrangement does not apply to swine. Consistent with weak Tdt expression in early sites of lymphogenesis, N‐region additions in VDJ rearrangements were more frequent at 95 DG. Junctional diversity in VλJλ rearrangement was limited at all stages of development. There was little evidence for B‐cell lymphogenesis in the ileal Peyers patches. The widespread recovery of VpreB transcripts in whole, non‐lymphoid tissue was unexpected as was its recovery from bone marrow and peripheral blood monocytes. Based on recovery of SJC, B‐cell lymphogenesis continues for at least 5 weeks postpartum.

Epitope Dampening Monotypic Measles Virus Hemagglutinin Glycoprotein Results in Resistance to Cocktail of Monoclonal Antibodies

The measles virus (MV) is serologically monotypic. Life-long immunity is conferred by a single attack of measles or following vaccination with the MV vaccine. This is contrary to viruses such as influenza, which readily develop resistance to the immune system and recur. A better understanding of factors that restrain MV to one serotype may allow us to predict if MV will remain monotypic in the future and influence the design of novel MV vaccines and therapeutics. MV hemagglutinin (H) glycoprotein, binds to cellular receptors and subsequently triggers the fusion (F) glycoprotein to fuse the virus into the cell. H is also the major target for neutralizing antibodies. To explore if MV remains monotypic due to a lack of plasticity of the H glycoprotein, we used the technology of Immune Dampening to generate viruses with rationally designed N-linked glycosylation sites and mutations in different epitopes and screened for viruses that escaped monoclonal antibodies (mAbs). We then combined rationally designed mutations with naturally selected mutations to generate a virus resistant to a cocktail of neutralizing mAbs targeting four different epitopes simultaneously. Two epitopes were protected by engineered N-linked glycosylations and two epitopes acquired escape mutations via two consecutive rounds of artificial selection in the presence of mAbs. Three of these epitopes were targeted by mAbs known to interfere with receptor binding. Results demonstrate that, within the epitopes analyzed, H can tolerate mutations in different residues and additional N-linked glycosylations to escape mAbs. Understanding the degree of change that H can tolerate is important as we follow its evolution in a host whose immunity is vaccine induced by genotype A strains instead of multiple genetically distinct wild-type MVs.

Immune response to recombinant visna virus Gag and Env precursor proteins synthesized in insect cells.

Two different recombinant visna virus (VV) gag-baculoviruses were constructed for the expression of precursor VV Gag in insect cells. Both recombinant Gag viruses expressed proteins migrating on SDS PAGE at the predicted rate for VV Gag precursor, Pr50gag. However, differences were seen in the morphology of the virus-like particles produced. Monoclonal antibody directed against the VV Gag capsid protein (p25) and sera from sheep infected with ovine lentiviruses reacted to both 50-kDa proteins. A recombinant VV env-baculovirus was constructed, substituting sequences encoding the signal peptide of VV Env with the murine IFN-gamma analogue. Sera from ovine lentivirus infected sheep reacted in immunoblots with two proteins of approximately 100 and 200 kDa found in the plasma membrane of insect cells infected with env-recombinant virus. Sheep immunized with either the recombinant Gag or the Env proteins developed high antibody titers to VV in ELISA. The serum of sheep and ascitic fluid of mice immunized with the recombinant Gag reacted with native Pr50gag and the processed Gag proteins in immunoblots, whereas serum of the recombinant Env immunized sheep reacted with VV gp135 and a putative oligomer of gp135. The immunized sheep responded specifically to visna virus by lymphocyte proliferation in vitro.

Synthesis and Assembly of Chimeric Human Immunodeficiency Virus Gag Pseudovirions

Expression of the HIV Gag precursor in insect cells by recombinant baculoviruses results in the assembly and budding of noninfectious pseudovirions that resemble immature virus. Three strategies for packaging additional viral epitopes into pseudovirions were examined: coinfection of insect cells with individual baculoviruses encoding separate Gag and Env structural genes, inframe Gag-Env fusion proteins, and Gag-frameshift-Env fusion proteins. Electron microscopy and Western blot analysis indicated that neither the coinfection nor the inframe fusion strategies reliably produced large quantities of structurally stable chimeric pseudovirions. The frameshift fusion method utilized the retroviral Gag-Pol ribosomal frameshift mechanism for the coexpression of Gag and Gag-frameshift-Env fusion proteins. Large quantities of pseudovirions containing both the Gag and Env epitopes were produced in insect cells. Mice inoculated with the Gag-frameshift-Env pseudovirions developed cytotoxic lymphocyte responses to both HIV Gag and Env epitopes. Vaccine and immunotherapeutic applications of chimeric pseudovirions are discussed.

Antibody repertoire development in fetal and neonatal piglets. XXIII: Fetal piglets infected with a vaccine strain of PRRS Virus display the same immune dysregulation seen in isolator piglets☆

The Ig levels and antibody repertoire diversification in fetal piglets infected with an attenuated Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) were measured. Serum Ig levels were greatly elevated in PRRSV-infected fetuses; IgG was elevated >50-fold, IgM>5-15-fold and IgA>2-fold compared to control fetuses. Their IgM to IgG to IgA profile was the same as that in isolator piglets infected for the same period with wild-type PRRSV. Fetal animals showed less repertoire diversification than even isolator piglets that were maintained germfree (GF) while the repertoire diversification index (RDI) for PRRSV-infected isolator piglets was 10-fold higher and comparable to littermates infected with swine influenza (S-FLU). However, when expressed as the RDI:Ig ratio, infected fetuses appeared 10-fold less capable of repertoire diversification than uninfected littermates and GF isolator piglets. Compared to S-FLU isolator piglets that resolve the infection, the RDI:Ig of PRRSV-infected isolator piglets was 100-fold lower. Overall, infection of fetuses with an attenuated virus shows the same immune dysregulation seen postnatally in wild type infected isolator piglets, indicating that: (a) attenuation did not alter the ability of the virus to cause dysregulation and (b) the isolator infectious model reflects the fetal disease.