Characterization of "Off-Target" Immune Modulation Induced by Live Attenuated Yellow Fever Vaccine.

BACKGROUND: Live attenuated vaccines alter immune functions and are associated with beneficial outcomes. We previously demonstrated that live attenuated yellow fever virus (YFV) vaccine (LA-YF-Vax) dampens T-cell receptor (TCR) signaling in vitro via an RNA-based mechanism. We examined study participants before and after LA-YF-Vax to assess TCR-mediated functions in vivo. METHODS: Serum samples and peripheral blood mononuclear cells (PBMCs) were obtained before and after LA-YF-Vax (with or without additional vaccines) or quadrivalent influenza vaccine. TCR-mediated activation was determined by interleukin 2 release or phosphorylation of the lymphocyte-specific Src kinase. TCR-regulating phosphatase (protein tyrosine phosphatase receptor type E [PTPRE]) expression was also measured. RESULTS: Compared with prevaccination findings, LA-YF-Vax recipient PBMCs demonstrated transient reduction in interleukin 2 release after TCR stimulation and PTPRE levels, unlike in control participants who received quadrivalent influenza vaccine. YFV was detected in 8 of 14 participants after LA-YF-Vax. After incubation of healthy donor PBMCs in serum-derived extracellular vesicles prepared from LA-YF-Vax recipients, TCR signaling and PTPRE levels were reduced after vaccination, even in participants without detectable YFV RNA. CONCLUSIONS: LA-YF-Vax reduces TCR functions and PTPRE levels after vaccination. Extracellular vesicles from serum recapitulated this effect in healthy cells. This likely contributes to the reduced immunogenicity for heterologous vaccines after LA-YF-Vax administration. Identification of specific immune mechanisms related to vaccines should contribute to understanding of the "off-target," beneficial effects of live vaccines.

Antigenic and immunogenic properties of recombinant hepatitis A virus 14S and 70S subviral particles.

Hepatitis A virus (HAV) has an immunodominant neutralization antigenic site. By using a panel of monoclonal antibodies targeted against the HAV neutralization antigenic site, it was shown that three epitopes within this site are present on 14S subunits (pentamers of the structural unit). In contrast, two other epitopes within this site are formed upon assembly of 14S subunits into capsids. Thus, the epitopes recognized by these two monoclonal antibodies are formed either by a conformational change in the antigenic site or by the juxtaposition of epitope fragments present on different 14S subunits during assembly of 14S into 70S particles. Both 14S and 70S particles elicited HAV-neutralizing antibodies in mice; thus, these particles may be useful for HAV vaccine development.

Expression of foreign epitopes on recombinant occlusion bodies of baculoviruses.

Recombinant polyhedrin proteins of the baculovirus Autographa californica nuclear polyhedrosis virus were constructed to serve as immunologic carriers of foreign epitopes. Several recombinants containing an influenza haemagglutinin epitope were obtained. Three of the five recombinants formed occlusion bodies (OBs) and two did not. All of the recombinant polyhedrin proteins reacted with a monoclonal antibody (mAb) specific for an influenza epitope in Western blots. Presentation of the foreign epitope on the surface of recombinant OBs was demonstrated by specific immunoprecipitation of the anti-influenza mAb with recombinant OBs. The recombinant polyhedrin-influenza epitope fusion protein stimulated an influenza-specific immune response in rabbits.

The hepatitis A virus polyprotein expressed by a recombinant vaccinia virus undergoes proteolytic processing and assembly into viruslike particles.

Hepatitis A virus (HAV) contains a single-stranded, plus-sense RNA genome with a single long open reading frame encoding a polyprotein of approximately 250 kDa. Viral structural proteins are generated by posttranslational proteolytic processing of this polyprotein. We constructed recombinant vaccinia viruses which expressed the HAV polyprotein (rV-ORF) and the P1 structural region (rV-P1). rV-ORF-infected cell lysates demonstrated that the polyprotein was cleaved into immunoreactive 29- and 33-kDa proteins which comigrated with HAV capsid proteins VP0 and VP1. The rV-P1 construct produced a 90-kDa protein which showed no evidence of posttranslational processing. Solid-phase radioimmunoassays with human polyclonal anti-HAV sera and with murine or human neutralizing monoclonal anti-HAV antibodies recognized the rV-ORF-infected cell lysates. Sucrose density gradients of rV-ORF-infected cell lysates contained peaks of HAV antigen with sedimentation coefficients of approximately 70S and 15S, similar to those of HAV empty capsids and pentamers. Immune electron microscopy also demonstrated the presence of viruslike particles in rV-ORF-infected cell lysates. Thus, the HAV polyprotein expressed by a recombinant vaccinia virus demonstrated posttranslational processing into mature capsid proteins which assembled into antigenic viruslike particles.

Cloning and Expression of an Insecticidal k-73 Type Crystal Protein Gene from Bacillus thuringiensis var. kurstaki into Escherichia coli.

A 75-kilobase plasmid from Bacillus thuringiensis var. kurstaki (HD-244) was associated with the k-73 type insecticidal crystal protein production by mating into B. cereus and subsequent curing of excess plasmids. This plasmid was partially digested with endonuclease R . Sau3A and the fragments were cloned into Escherichia coli (HB101) on vector pBR322. Candidate clones were screened for plasmid vectors which contained the expected insert size (at least 3 kilobases) and then with an enzyme-linked immunosorbent assay, using antisera prepared against electrophoretically purified, solubilized insecticidal crystal protein of 130,000 daltons. Several positive clones were isolated and were analyzed for expression, toxicity, and genetic content by restriction enzyme analysis. Electrophoretic transfer blots of proteins from a candidate E. coli clone, analyzed by enzyme-linked immunosorbent assay, demonstrated a predominant cross-reacting protein of about 140,000 daltons. Ouchterlony analysis also showed a single precipitin band. Extensive bioassays with Manduca sexta larvae revealed that the E. coli clones make toxin with a specific activity (50% lethal dose per microgram of cross-reacting protein) equivalent to that of the parental B. thuringiensis strain or a B. cereus trancipient carrying the toxin-encoding, 75-kilobase plasmid.

Conformational studies on the inactivation of glyceraldehyde-3-phosphate dehydrogenase from the facultative thermophile Bacillus coagulans KU.

Among the various proposals that have been made in attempting to explain the ability of thermophiles to reproduce at high temperatures, there is no doubt that obligate and extreme thermophiles synthesize proteins (and other molecules) that have sufficient intrinsic molecular stability to withstand increased thermal stress. In contrast, the glyceraldehyde-3-phosphate dehydrogenase from the facultative thermophile Bacillus coagulans KU has been shown to be quite thermolabile in vitro. Thermal inactivation is not due to loss of bound NAD+. It has also been shown that the enzymatic activity can be thermostabilized in vitro by increased ionic strength. As previously reported [J. W. Crabb, A. L. Murdock, and R. E. Amelunxen (1975) Biochem. Biophys. Res. Commun. 62, 627; (1977) Biochemistry 16, 4840], the enzyme loses 94-97% of enzymatic activity after heat treatment at 55 degrees C for 5 min in 0.05 M sodium phosphate buffer (pH 7.1); however, by increasing the ionic strength to 1.8, complete protection was conferred at this temperature. Gel-filtration chromatography has been used to study the initial dissociation and subsequent aggregation of the glyceraldehyde-3-phosphate dehydrogenase after thermal inactivation. Aggregation occurs when the enzyme is heated at 50 degrees or 55 degrees C. Loss of enzymatic activity is correlated with changes in the tertiary structure as measured by the near-uv CD spectrum of the enzyme following heat inactivation, with essential disappearance of the peaks at 263 and 296 nm, and a blue shift of the far-uv spectrum, which is a measure of secondary structure. Estimation of secondary structure of the unheated protein from the far-uv CD data showed the enzyme contains approximately 26% alpha-helix, approximately 21% beta-structure, and approximately 53% disordered structure. Heat treatment at various temperatures resulted in only slight changes of the estimated secondary structure. Increased ionic strength prevents thermal alteration of the CD spectrum in both near- and far-uv regions. The data support the previous proposal that thermolabile enzymes such as the glyceraldehyde-3-phosphate dehydrogenase from the facultative thermophile B. coagulans are thermostabilized in vivo mainly by the intracellular charged macromolecular environment.

Sequence homology in the amino-terminal and active-site regions of thermolabile glyceraldehyde-3-phosphate dehydrogenase from a thermophile.

The unusual thermolability of glyceraldehyde-3-phosphate dehydrogenase from the facultative thermophile Bacillus coagulans KU (Crabb et al., Biochemistry 16:4840-4847, 1977) has provided the first opportunity to study a homologous enzyme from the same genus that exhibits a marked difference in thermostability. In pursuit of the structural bases for the thermostability of proteins, the sequences of the amino terminus (residues 1 through 27) and the active-site cysteine cyanogen bromide peptide (residues 130 through 167) of this enzyme have been determined and compared with sequences of the enzyme from other sources. The importance of comparing phylogenetically related proteins is evident from the 87% identity found between these sequences in the enzyme from B. coagulans and Bacillus stearothermophilus, versus only 45% identity for all other known sequences. The marked sequence identity of the enzyme from the two Bacillus species drew attention to the variable region (residues 138 through 140a) which is exposed to the exterior of the quaternary structure of this enzyme. Based on the reported crystallographic structures of the enzyme from lobster muscle and B. stearothermophilus and space-filling models of the variable region, the segment Asp-Pro-Lys-Ala in B. stearothermophilus should be more thermostable than the analogous sequence, Asp-Ala-Ala-Asn, from B. coagulans. In addition, the space-filling models suggested that the spatial relationship of an amino acid side chain and its potential for close packing and interactions with neighboring side chains may be more important than the type of amino acid substituted.