Carbohydrate biopolymers enhance antibody responses to mucosally delivered vaccine antigens.

We have evaluated the ability of two carbohydrate biopolymers, chitosan and gellan, to enhance antibody responses to subunit influenza virus vaccines delivered to the respiratory tracts of mice. Groups of mice were vaccinated three times intranasally (i.n.) with 10 microg of purified influenza B/Panama virus surface antigens (PSAs), which consist of hemagglutinin (HA) and neuraminidase (NA), either alone or admixed with chitosan or gellan solutions. Separate groups were vaccinated subcutaneously (s.c.) with PSAs adsorbed to Alhydrogel or chitosan or gellan alone i.n. Serum antibody responses were determined by enzyme-linked immunosorbent assay (ELISA) for influenza virus-specific immunoglobulin G (IgG) and by HA inhibition (HAI) and NA inhibition (NAI) assays. The local respiratory immune response was measured by assaying for influenza virus-specific IgA antibody in nasal secretions and by enumerating nasal and pulmonary lymphocytes secreting IgA, IgG, and IgM anti-influenza virus-specific antibodies by enzyme-linked immunospotting (ELISPOT). When administered alone i.n., B/Panama PSA was poorly immunogenic. Parenteral immunization with B/Panama PSA with Alhydrogel elicited high titers of anti-B/Panama antibodies in serum but a very poor respiratory anti-B/Panama IgA response. In contrast, i.n. immunization with PSA plus chitosan stimulated very strong local and systemic anti-B/Panama responses. Gellan also enhanced the local and serum antibody responses to i.n. PSA but not to the same extent as chitosan. The ability of chitosan to augment the immunogenicity of influenza vaccines given i.n. was confirmed using PSA prepared from an influenza A virus (A/Texas H1N1).

Studies on the IgA-independent immunological responses in mice to influenza virus challenge after oral vaccination with irradiated whole virus and an erythrocyte complex.

Previous studies have described an oral influenza vaccine comprising whole irradiated virus and an erythrocyte complex (IV-EC), which gave broad-based protection against influenza virus challenge in mice. The present study examined the immune responses generated after live virus challenge of vaccinated mice, particularly to determine whether mice vaccinated with IV-EC had enhanced CTL activity to compensate for the previously reported diminution in lung IgA response. Oral vaccine groups examined were IV-EC, live virus alone (LV) or live virus-erythrocyte complex (LV-EC), compared with irradiated virus and erythrocyte alone controls. The antibody responses of IV-EC and LV-EC vaccinated mice showed significantly elevated lung and serum IgG2a levels post live virus challenge, with no comparable increases in IgG1 levels compared to controls. Spleen cells from IV-EC mice showed an enhanced post-challenge proliferative response to antigen compared with mice that had received live oral vaccines, indicating enhanced cellular activity post IV-EC immunization. However, CTL activity was not enhanced for IV-EC mice, and live virus-vaccinated mice had reduced CTL activity compared with controls, indicating that CTL were not important for post-vaccine protection. Cytokine analysis revealed a predominant IFN-gamma response in spleen cells from orally vaccinated mice, whereas IL-4 was not detected in any lung or spleen culture analysed. The results suggest, therefore, that protection from live influenza challenge after IV-EC or LV-EC vaccination was due to an IFN-mediated IgG2a response. Definitive confirmation of the role of these factors in post-vaccine protection can now be tested in IgG2a-depleted or IFN-gamma gene knockout mouse models.

Erythrocytes enhance the immunogenicity of oral vaccination with gamma irradiated influenza virus: increasing the dose of irradiation results in a significant diminution of lung IgA response.

Previous studies have demonstrated that the ability of gamma-irradiated whole influenza virus to prime for specific anti-influenza antibody responses was dramatically enhanced when delivered in a complex with chicken red blood cell ghosts (cRBC). The purpose of this study was to investigate the effect of increasing the dose of gamma irradiation used to inactivate A/Queensland/6/72 virus on the ability of the virus-cRBC complex to prime for specific influenza responses. Spleen cell proliferation studies confirmed the enhancing effect of the cRBC carrier for oral vaccination with irradiated virus. Cells from mice vaccinated with 30 kGy-irradiated virus only did not respond to influenza stimulation in vitro, whereas cells from mice vaccinated with irradiated virus+cRBC showed significant increases in proliferation to antigen exposure. No significant antibody response or challenge virus clearance was observed in mice orally vaccinated with irradiated (13.1 or 30 kGy) virus alone, even when the dose was increased significantly. Oral vaccination with live virus (+/-cRBC) primed for significant influenza specific IgA responses in the lungs, in addition to IgG responses in the lungs and sera. The dose of irradiation used to inactivate the virus was found to be critical to the profile of antibody response when the virus was delivered in a complex with cRBC. Oral vaccination of Swiss mice with 13.1 or 30 kGy virus (+cRBC) primed for significant serum and lung IgG responses. Lung IgA responses for 13.1 kGy+cRBC vaccinated mice were detected, but 30 kGy+cRBC vaccinated Swiss and CBA/H mice had no significant lung IgA response. The abrogation of IgA response, however, did not lessen the clearance of live challenge virus in outbred mice, suggesting a primary role for IgG and/or CTL response in the control of influenza virus infection post oral vaccination. To ensure direct comparison of virus alone and virus+cRBC treatments, the concentration of virus complexed to the cRBC was determined.

X-ray solution scattering of Sindbis virus. Changes in conformation induced at low pH.

Alphaviruses, like many enveloped animal viruses, enter the cell by fusing with the cell membrane. This fusion occurs only in coated vesicles at a low pH. By using X-ray solution scattering of highly purified virus particles we have gained direct evidence that a drop in pH does not alter the structure of the virus core but does cause a significant change in the structure of the virus envelope. Thus these experiments give direct evidence to support the hypothesis that a reduction in pH causes a conformational change in the virus E protein, which enables it to promote fusion with the cell envelope and trigger virus infection.

Plasma fibronectin prepared from patients with metastatic breast cancer shows in vitro aggregation property.

Plasma fibronectin purified from the plasma of metastatic breast cancer patients has been studied by light scattering. It clearly shows abnormal self-aggregation properties; the possible significance of these findings to the in vivo situation is discussed.

Functional reconstitution of the integral membrane proteins of influenza virus into phospholipid liposomes.

The integral membrane proteins of influenza virus, a hemagglutinin and a neuraminidase, have been incorporated into liposomes composed of either phosphatidylcholine or a mixture of phosphatidylcholine and phosphatidylethanolamine (2:1 w/w) using detergent dialysis. The virus spike glycoproteins for reconstitution were selectively solubilized by using cetyltrimethylammonium bromide to leave a "core particle", which lacked a lipid bilayer but possessed quaternary structure as observed by electron microscopy. The viral spike proteins were combined with exogenous phospholipid in excess sodium cholate followed by exhaustive dialysis for 150 h. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that only the viral glycoproteins were associated with all the complexes formed. The level of sodium cholate remaining after dialysis was shown to be reduced to less than 1 molecule per 80 protein molecules. Viral proteins reconstituted into dimyristoylphosphatidylcholine liposomes were shown to have retained hemagglutination, low-pH-dependent hemolysis, and neuraminidase activities and were associated with a lipid bilayer in two types of complexes with average lipid to protein mole ratios after sucrose density gradient purification of either 590:1 or 970:1. The bilayer vesicles formed were of similar sizes and were shown by negative-stain electron microscopy to be 150-300 nm in diameter with well-defined spikes on their surface. Reconstituted liposomes of dimyristoylphosphatidylcholine were found to be unstable with respect to their trapped volume and therefore were unsuitable for fusion studies, unlike complexes formed with phosphatidylcholine or a mixture of phosphatidylcholine/phosphatidylethanolamine derived from hen eggs.(ABSTRACT TRUNCATED AT 250 WORDS)