Serum antibody responses in children with rotavirus diarrhea can serve as proxy for protection.

We examined sera from 42 patients 1 to 30 months of age for rotavirus immunoglobulin M (IgM), IgA, IgG, and IgG subclasses and sought to determine if serum antibody could serve as a reliable marker for prediction of disease severity. Infants in the first few months of life usually had high maternal IgG titers and, when they were infected with rotavirus, had low IgM titers or no IgM in acute-phase sera and poor seroconversions 3 weeks later, suggesting that maternal antibodies had inhibited viral replication and antibody responses. All patients > or =6 months of age had IgM in acute-phase sera, indicating that IgM is a good marker for acute rotavirus infection. IgG was the best overall predictor of an infection, as the convalescent-phase sera of 81% of the patients had a fourfold rise in the IgG titer. IgA titers in convalescent-phase sera and conversion rates were higher among patients > or =12 months of age than among children younger than 12 months. IgG1 was the predominant subclass detected in the acute-phase sera of some children and in all 28 convalescent-phase serum samples examined. Patients with preexisting acute-phase IgG titers of > or =100 or > or =200 had diarrhea that was less severe or of a shorter duration. These results indicate that serum IgG is the most reliable marker for seroconversion and is a consistent proxy for protection against severe disease.

Induction of tumor-specific immunity in mice by immunization with reconstituted tumor membrane liposomes containing recombinant B7-2 (CD86).

There has been considerable interest in developing experimental vaccines using genetically modified tumor cells expressing cytokines or costimulatory molecules to enhance immunogenicity. The authors investigated an alternative approach of using protein transfer rather than gene transfer to introduce costimulatory molecules rapidly into tumor membranes. Immunization with a single dose of reconstituted tumor membrane liposomes containing purified recombinant B7-2 (CD86) induced tumor rejection in mice challenged with syngeneic tumors, including the poorly immunogenic AG104A fibrosarcoma. These findings support the possibility that cell-free vaccines composed of reconstituted tumor membrane liposomes containing additional immunostimulatory proteins may offer a practical and safe alternative to genetically modified tumor cells for treating human cancer.

Protein transfer of the costimulatory molecule, B7-2 (CD86), into tumor membrane liposomes as a novel cell-free vaccine.

Several approaches have been taken to enhance the immunogenicity of tumors. Genetically-modified tumors expressing various cytokines, major histocompatibility complex (MHC) molecules, or costimulatory molecules such as B7-1 (CD80) or B7-2 (CD86) can induce tumor-specific immune responses. In the present study, an alternative approach was explored based on direct protein transfer of purified recombinant B7-2 into tumor cell membranes. B7-2 was purified from recombinant baculovirus infected insect cells. Although differentially glycosolyated, the recombinant B7-2 retained the function to costimulate T-cell proliferation. Purified B7-2 was readily incorporated into tumor membranes using a detergent dialysis technique to form unilameller liposomes. The immunogenicity of tumor membrane proteoliposomes was significantly increased by incorporation of B7-2. These findings suggest an alternative method for the introduction of immunostimulatory molecules into tumor membranes to create novel tumor vaccines.

A herpes simplex virus 1 recombinant lacking the glycoprotein G coding sequences is defective in entry through apical surfaces of polarized epithelial cells in culture and in vivo.

During infection of a new host, the first surfaces encountered by herpes simplex viruses are the apical membranes of epithelial cells of mucosal surfaces. These cells are highly polarized, and the protein composition of their apical and basolateral membranes are very different, so that different viral entry pathways have evolved for each surface. To determine whether the viral glycoprotein G (gG) is specifically required for efficient infection of a particular surface of polarized cells, apical and basal surfaces were infected with wild-type virus or a gG deletion mutant. After infection of polarized cells in culture, the gG(-) virus was deficient in infection of apical surfaces but was able to infect cells through basal membranes, replicate, and spread into surrounding cells. The gG-dependent step in apical infection was a stage beyond attachment. After in vivo infection of apical surfaces of epithelial cells of nonscarified mouse corneas, infection by glycoprotein C(-) or gG(-) virus was considerably reduced as compared with that observed after infection with wild-type virus. In contrast, when corneas were scarified, allowing virus access to other cell surfaces, the gG and glycoprotein C deletion mutants infected eyes as efficiently as wild-type viruses. A secondary mutation allowing infection of apical surfaces by gG(-) virus arose readily during passage of the virus in nonpolarized cells, indicating that either the gG-dependent step of apical infection can be bypassed or that another viral protein can acquire the same function.

Peptide exchange in MHC molecules.

Major histocompatibility complex (MHC)-encoded glycoproteins bind peptide antigens through non-covalent interactions to generate complexes that are displayed on the surface of antigen-presenting cells (APC) for recognition by T cells. Peptide-binding site occupancy is necessary for stable assembly of newly synthesized MHC proteins and export from the endoplasmic reticulum (ER). The MHC class II antigen-processing pathway provides a mechanism for presentation of peptides generated in the endosomal pathway of APC. The chaperone protein, invariant chain, includes a surrogate peptide that stabilizes newly synthesized class II molecules during transport to endosomal compartments. The invariant chain-derived peptide must be replaced through a peptide exchange reaction that is promoted by acidic pH and the MHC-encoded co-factor HLA-DM. Peptide exchange reactions are not required for presentation of antigens by MHC class I molecules because they bind antigens during initial assembly in the ER. However, exchange reactions may play an important role in editing the repertoire of peptides presented by both class II and class I molecules, thus influencing the specificity of immunity and tolerance.

Intact proteins can bind to class II histocompatibility molecules with high affinity.

The ability of intact protein antigens to bind to purified class II histocompatibility molecules was investigated. Intact bovine ribonuclease (RNase) inhibited peptide binding to DR1 with a potency similar to that of a high affinity peptide or irreversibly denatured RNase. Similarly, horse myoglobin (Mb) was a potent inhibitor of peptide binding to I-E(k). I-E(k)-Mb complexes were directly visualized as a distinct band with reduced mobility on SDS PAGE. Direct binding experiments with biotin-labeled proteins demonstrated that Mb and RNase bind to class II molecules through the peptide-binding groove with high affinity, and that binding occurs in the absence of detergent. The possibility that HLA-DM can catalyse the binding of intact protein antigens was supported by the observation that DM enhances the binding of biotin-RNase to DR1. Our results provide further support for the hypothesis that intact, partially unfolded protein antigens can act as ligands for initial interaction with class II molecules.