Human Cytomegalovirus (HCMV)-Specific Antibody Response and Development of Antibody-Dependent Cellular Cytotoxicity Against HCMV After Lung Transplantation.

BACKGROUND: Human cytomegalovirus (HCMV) may cause severe infections in lung transplant recipients (LTRs). The impact of the host antibody (AB)-dependent cytotoxicity (ADCC) on HCMV is still unclear. Therefore, we analyzed the AB-response against HCMV glycoprotein B (gB) and the pentameric complex (PC) and the ADCC response in HCMV-seropositive (R+) LTRs and in seronegative recipients of positive organs (D+/R-). METHODS: Plasma samples were collected from 35 R+ and 28 D+/R- LTRs for 1 (R+) or 2 (D+/R-) years posttransplantation and from 114 healthy control persons. The PC- and gB-specific ABs were assessed by enzyme-linked immunosorbent assay. The ADCC was analyzed by focal expansion assay and CD107 cytotoxicity assay. RESULTS: In R+ LTRs, significantly higher gB-specific AB levels developed within 1 year posttransplantation than in controls (immunoglobulin [Ig]G1, P < .001; IgG3, P < .001). In addition, higher levels of ADCC were observed by FEA and CD107 assay in R+ patients compared with controls (P < .001). In 23 D+R- patients, HCMV-specific ABs developed. Antibody-dependent cytotoxicity became detectable 3 months posttransplantation in these, with higher ADCC observed in viremic patients. Depletion of gB- and PC-specific ABs revealed that, in particular, gB-specific Abs were associated with the ADCC response. CONCLUSIONS: We show that a strong ADCC is elicited after transplantation and is especially based on gB-specific ABs.

Two classes of protective antibodies against Pseudorabies virus variant glycoprotein B: Implications for vaccine design.

Pseudorabies virus (PRV) belongs to the Herpesviridae family, and is an important veterinary pathogen. Highly pathogenic PRV variants have caused severe epidemics in China since 2011, causing huge economic losses. To tackle the epidemics, we identified a panel of mouse monoclonal antibodies (mAbs) against PRV glycoprotein B (gB) that effectively block PRV infection. Among these 15 mAbs, fourteen of them block PRV entry in a complement-dependent manner. The remaining one, 1H1 mAb, however can directly neutralize the virus independent of complement and displays broad-spectrum neutralizing activities. We further determined the crystal structure of PRV gB and mapped the epitopes of these antibodies on the structure. Interestingly, all the complement-dependent neutralizing antibodies bind gB at the crown region (domain IV). In contrast, the epitope of 1H1 mAb is located at the bottom of domain I, which includes the fusion loops, indicating 1H1 mAb might neutralize the virus by interfering with the membrane fusion process. Our studies demonstrate that gB contains multiple B-cell epitopes in its crown and base regions and that antibodies targeting different epitopes block virus infection through different mechanisms. These findings would provide important clues for antiviral drug design and vaccine development.

Allotype analysis to determine the origin of cytomegalovirus immunoglobulin-G after allogeneic stem cell transplantation.

BACKGROUND: Cytomegalovirus (CMV) reactivation still remains a major problem following allogeneic hematopoietic stem cell transplantation (HSCT). PATIENTS AND METHODS: In this study, we analyzed an immunoglobulin allotype, IgG1m(f), in CMV-seropositive HSCT recipients and their donors to distinguish donor-derived antibody from recipient-derived antibody. Eight donor-recipient pairs were informative regarding the appearance of donor-derived immunoglobulin-G (IgG), as the recipients were homozygous null for the IgG1m(f) allotype and the donors were IgG1m(f) positive. In these patients, total IgG, IgM, and allotype-specific IgG against CMV were measured by enzyme-linked immunosorbent assay. All subjects were monitored for at least 9 months after HSCT with (n = 5) or without (n = 3) CMV reactivation. RESULTS: Donor-derived CMV IgG tended to be elevated earlier in patients with CMV-seropositive donors than in those with CMV-seronegative donors. In 1 patient with a CMV-negative donor, donor-derived CMV IgG was not detected until late CMV reactivation. In 3 patients without CMV reactivation, donor-derived CMV IgG was also elevated within 1-6 months after HSCT. CONCLUSION: In conclusion, the CMV serostatus of the donor may be related to the timing of the appearance of donor-derived CMV IgG and the reconstitution of humoral immunity against CMV, regardless of the CMV antigenemia level after HSCT.

Medical devices; immunology and microbiology devices; classification of John Cunningham Virus serological reagents. Final order.

The Food and Drug Administration (FDA) is classifying John Cunningham Virus (JCV) serological reagents into class II (special controls). The Agency is classifying the device into class II (special controls) in order to provide a reasonable assurance of safety and effectiveness of the device.

Single domain antibody multimers confer protection against rabies infection.

Post-exposure prophylactic (PEP) neutralizing antibodies against Rabies are the most effective way to prevent infection-related fatality. The outer envelope glycoprotein of the Rabies virus (RABV) is the most significant surface antigen for generating virus-neutralizing antibodies. The small size and uncompromised functional specificity of single domain antibodies (sdAbs) can be exploited in the fields of experimental therapeutic applications for infectious diseases through formatting flexibilities to increase their avidity towards target antigens. In this study, we used phage display technique to select and identify sdAbs that were specific for the RABV glycoprotein from a naïve llama-derived antibody library. To increase their neutralizing potencies, the sdAbs were fused with a coiled-coil peptide derived from the human cartilage oligomeric matrix protein (COMP48) to form homogenous pentavalent multimers, known as combodies. Compared to monovalent sdAbs, the combodies, namely 26424 and 26434, exhibited high avidity and were able to neutralize 85-fold higher input of RABV (CVS-11 strain) pseudotypes in vitro, as a result of multimerization, while retaining their specificities for target antigen. 26424 and 26434 were capable of neutralizing CVS-11 pseudotypes in vitro by 90-95% as compared to human rabies immunoglobulin (HRIG), currently used for PEP in Rabies. The multimeric sdAbs were also demonstrated to be partially protective for mice that were infected with lethal doses of rabies virus in vivo. The results demonstrate that the combodies could be valuable tools in understanding viral mechanisms, diagnosis and possible anti-viral candidate for RABV infection.

Relationship of serological subtype, basic core promoter and precore mutations to genotypes/subgenotypes of hepatitis B virus.

Using phylogenetic analysis and pairwise comparison of 670 complete hepatitis B virus (HBV) genomes, we demonstrated that nucleotide divergence greater than 7.5% can be used to separate strains into genotypes A-H. Strains can be separated into subgenotypes when two criteria are met: nucleotide divergence of about 4% but less than 7.5% and good bootstrap support. There is a highly statistically significant association between serological subtypes and genotypes (chi2-test for association, P < 0.0001): adw is associated with genotypes A, B, F, G, and H, adr with C and ayw with D and E. The logistic regression method showed that 1802-1803CG are characteristic of genotypes A, D, and E whereas 1802-1803TT are characteristic of genotypes B, C, and F. 1858C is positively associated with genotypes A, F, and H and 1858T with genotypes B, D, and E. Subgenotypes C2, F1/F4 can be differentiated from subgenotypes C1, F2/F3, respectively, because the latter have 1858C as opposed to 1858T in the former. 1888A was positively associated with subgenotype A1 and TAA at 1817 with genotype G. The Haploplot method revealed high linkage between loci 1858 and 1896 but strong evidence of recombination between loci 1862 and 1896. Loci 1809-1812, 1862, and 1888 may have co-evolved. Using a computer program, we showed that serological subtype deduced from the S region (position 155-835) and mutations/variations within the basic core promoter/precore region (1653-1900), allowed genotyping of HBV with 97% sensitivity and 99% specificity. Certain subgenotypes or subgenotype groups could also be differentiated.

Immunity against infectious diseases: predictive value of self-reported history of vaccination and disease.

OBJECTIVE: To determine whether self-reported history of disease and/or vaccination is predictive of immunity against hepatitis B, varicella, rubella, mumps, and measles. DESIGN: The seroprevalence of viral antibodies and the predictive value of a self-report questionnaire were determined for 616 paramedical students who matriculated into Padua Medical School (Padua, Italy) during 2003-2005. RESULTS: The majority of subjects (86.9%) remembered being vaccinated against hepatitis B but had no recollection of disease. Among vaccinees, 1.5% showed markers of previous infection, 6.7% tested negative for anti-hepatitis B virus surface antigen (anti-HBsAg) antibodies, and 91.8% tested positive for anti-HBsAg. Self-reported vaccination history had a positive predictive value of 93.2% for test results positive for immunity against hepatitis B. Immunity against varicella (93.7% of subjects) and rubella (95.5%) was high, compared with immunity against mumps (79.9%) and measles (83.1%). In addition, results of tests for detection of immunity against mumps and measles were equivocal for more than 7% of subjects, probably because their vaccination regimen was not completed. Self-reported histories of varicella disease and rubella disease and vaccination had high positive predictive values (greater than 98% each) for testing positive for antiviral antibodies, compared with self-reported histories of mumps disease and vaccination and measles disease and vaccination; however, high positive predictive values were observed for self-reported histories of mumps only (92.0%) and measles only (94.7%). CONCLUSIONS: The self-report questionnaire used in this study did not accurately predict immunity against 5 transmittable but vaccine-preventable diseases. A complete serological evaluation of healthcare workers, followed by vaccination of those with negative or equivocal results of serological tests, is an appropriate measure to decrease the risk of infection in this population.

Neonatal immunization with a Sindbis virus-DNA measles vaccine induces adult-like neutralizing antibodies and cell-mediated immunity in the presence of maternal antibodies.

Infants younger than age 9 mo do not respond reliably to the live attenuated measles vaccine due the immaturity of their immune system and the presence of maternal Abs that interfere with successful immunization. We evaluated the immune responses elicited by Sindbis virus replicon-based DNA vaccines encoding measles virus (MV) hemagglutinin (H, pMSIN-H) or both hemagglutinin and fusion (F, pMSINH-FdU) glycoproteins in neonatal mice born to naive and measles-immune mothers. Despite the presence of high levels of maternal Abs, neonatal immunization with pMSIN-H induced long-lasting, high-avidity MV plaque reduction neutralization (PRN) Abs, mainly IgG2a, that also inhibited syncytium formation in CD150(+) B95-8 cells. IgG secreting plasma cells were detected in spleen and bone marrow. Newborns vaccinated with pMSINH-FdU elicited PRN titers that surpassed the protective level (200 mIU/ml) but were short-lived, had low syncytium inhibition capacity, and lacked avidity maturation. This vaccine failed to induce significant PRN titers in the presence of placentally transferred Abs. Both pMSIN-H and pMSINH-FdU elicited strong Th1 type cell-mediated immunity, measured by T cell proliferation and IFN-gamma production, that was unaffected by maternal Abs. Newborns responded to measles DNA vaccines with similar or even higher PRN titers and cell-mediated immunity than adult mice. This study is the first demonstration that a Sindbis virus-based measles DNA vaccine can elicit robust MV immunity in neonates bypassing maternal Abs. Such a vaccine could be followed by the current live attenuated MV vaccine in a heterologous prime-boost to protect against measles early in life.

Characterization and application of monoclonal antibodies against turbot (Scophthalmus maximus) Rhabdovirus.

Five monoclonal antibodies (mAbs), 1G8, 1H9, 2D2, 2D3, and 2F5, against Scophthalmus maximus rhabdovirus (SMRV) were prepared. Characterization of the mAbs included indirect enzyme-linked immunosorbent assay, isotyping, viral inhibition assay, immunofluorescence staining of virus-infected cell cultures, and Western blot analysis. Isotyping revealed that 1G8 and 1H9 were of the IgG2b subclass and that the other three were IgM. 2D2, 2D3, and 2F5 partially inhibited SMRV infection in epithelioma papulosum cyprinid (EPC) cell culture. Western blotting showed that all five mAbs could react with two SMRV proteins with molecular masses of approximately 30 kDa (P) and 26 kDa (M). These two proteins were localized within the cytoplasm of SMRV-infected EPC cells by immunofluorescence assay. Also, progressive foci of viral replication in cell cultures were monitored from 6 to 24 h, using mAb 2D3 as the primary antibody. A flow cytometry procedure was used to detect and quantify SMRV-infected (0.01 PFU/cell) EPC cells with mAb 2D3, and 10.8% of cells could be distinguished as infected 36 h postinfection. Moreover, mAb 2D3 was successfully applied for the detection of viral antigen in cryosections from flounder tissues by immunohistochemistry tests.

Development of a biosensor-based method for detection and isotyping of antibody responses to adenoviral-based gene therapy vectors.

A biosensor-based assay, using a surface plasmon resonance detection system, was developed to detect and isotype anti-adenoviral antibodies in patients dosed with an adenoviral-based gene therapy vector. In the assay, whole, intact virus was immobilized onto the sensor chip surface. Electron microscopy and monoclonal antibody studies provide evidence that the virus remains intact after immobilization. The patients tested had preexisting serum levels of anti-adenoviral antibodies. A classic anamnestic response was observed in patients dosed with the gene-therapy agent. Isotyping experiments indicated that IgG antibodies predominated in serum even at the predose time point. Analysis of ascites fluid samples from some patients indicated detectable levels of IgA in addition to IgG. Results obtained using the biosensor-based assay corresponded to an existing enzyme-linked immunosorbent assay. The assay was easy to perform and the automated instrument reduced the required "hands on" time. In addition to studying the development of anti-adenoviral antibodies, the techniques described may be applied to virus:receptor interaction studies or antiviral drug:virus interaction studies.

Human papillomavirus virus-like particles are efficient oral immunogens when coadministered with Escherichia coli heat-labile enterotoxin mutant R192G or CpG DNA.

Certain human papillomaviruses (HPVs) cause most cervical cancer, which remains a significant source of morbidity and mortality among women worldwide. HPV recombinant virus-like particles (VLPs) are promising vaccine candidates for controlling anogenital HPV disease and are now being evaluated as a parenteral vaccine modality in human subjects. Vaccines formulated for injection generally are more costly, more difficult to administer, and less acceptable to recipients than are mucosally administered vaccines. Since oral delivery represents an attractive alternative to parenteral injection for large-scale human vaccination, the oral immunogenicity of HPV type 11 (HPV-11) VLPs in mice was previously investigated; it was found that a modest systemic neutralizing antibody response was induced (R. C. Rose, C. Lane, S. Wilson, J. A. Suzich, E. Rybicki, and A. L. Williamson, Vaccine 17:2129-2135, 1999). Here we examine whether VLPs of other genotypes may also be immunogenic when administered orally and whether mucosal adjuvants can be used to enhance VLP oral immunogenicity. We show that HPV-16 and HPV-18 VLPs are immunogenic when administered orally and that oral coadministration of these antigens with Escherichia coli heat-labile enterotoxin (LT) mutant R192G (LT R192G) or CpG DNA can significantly improve anti-VLP humoral responses in peripheral blood and in genital mucosal secretions. Our results also suggest that LT R192G may be superior to CpG DNA in this ability. These findings support the concept of oral immunization against anogenital HPV disease and suggest that clinical studies involving this approach may be warranted.

Oral vaccination of mice with human papillomavirus virus-like particles induces systemic virus-neutralizing antibodies.

To assess whether oral vaccination against human papillomavirus (HPV) may be feasible, we administered HPV virus-like particles (VLPs) to mice by gavage. Enzyme-linked immunosorbent assay (ELISA) results indicated that serum anti-VLP immunoglobulin G (IgG) and IgA antibodies were induced after oral vaccination, and these responses demonstrated antigenic specificities that were conformationally dependent and restricted according to HPV genotype. Importantly, orally induced postimmune sera were found to neutralize HPV-11 virions in vitro. These results indicated that the VLPs were antigenically stable in the environment of the gastrointestinal tract and were able to engage in potentially useful immune system interactions. These findings support the concept of oral vaccination against anogenital HPV disease, and suggest the possibility that this may be a useful approach to the immunization of large populations against cervical cancer and other HPV associated diseases.

Increased levels of soluble tumour necrosis factor receptor-I (P55) and decreased IgG1 reactivities in HIV-1 patients with cytomegalovirus disease.

The purpose of the study was to investigate potential associations between tumour necrosis factor (TNF), soluble TNF receptors (sTNF-Rs), immunoglobulin (Ig)G subclasses and development of cytomegalovirus (CMV) disease amongst human immunodeficiency virus (HIV)-1 patients. We enrolled HIV-1 patients with CD4 counts less than 100/microl in a prospective study and followed them over 1 year for development of CMV disease. Concentrations of TNF, sTNF-RI, sTNF-RII and IgG subclass reactivities were measured by ELISA; levels of CMV pp65 antigenaemia were determined as numbers of pp65 expressing cells/100,000 cells and were measured by staining of leucocytes; and HIV-1 RNA loads were measured by polymerase chain reaction (PCR). Eighteen patients studied with CMV disease had higher levels of sTNF-RI than 18 similar patients without CMV disease. Concentrations of sTNF-RI correlated with levels of CMV antigenaemia in blood samples collected before the development of CMV disease. Patients with CMV disease had lower levels of IgG1 reactivities to CMV than patients without CMV disease. We conclude that increased levels of sTNF-RI and decreased IgG1 reactivities are associated with an increased risk of development of CMV disease among HIV-1 patients.

A serological indication of the existence of a guineapig poliovirus.

Attempts were made to clarify whether laboratory guineapigs may harbour a poliovirus which, in 1911, was described as the cause of a disease called guineapig lameness. By the use of ELISA for antibodies against the poliovirus, Theiler's murine encephalomyelitis virus (TMEV), it was shown that two pet shop guineapigs suffering from lameness had extremely high titres against poliovirus, while healthy guineapigs from the same pet shop were negative. Clearly positive results were also found in 35 out of 152 laboratory guineapig sera. Positive results were found in only two out of six breeding centres, but in three out of three experimental units, all of which purchased guineapigs from one of the seropositive breeding colonies. The diseased guineapigs recovered fully after treatment with vitamins in the drinking water, a treatment used for guineapig lameness by small animal practitioners. A theory that vitamin C deficient guineapigs are, due to an impaired steroid secretion, predisposed to succumbing to infection and develop demyelinating disease similar to that in TMEV infected mice is discussed briefly. Guineapig sera were also tested serologically for other infections. Antibodies against lymphocytic choriomeningitis virus, Clostridium piliforme and Toxoplasma gondii were not found, but one breeding colony was infected with adenovirus, pneumonia virus of mice, reovirus type 3, Sendai virus, parainfluenza (simian) virus type 5 and Encephalitozoon cuniculi. Two other breeding colonies were infected with both reovirus type 3 and E. cuniculi. In all three experimental units infection with adenovirus was observed, and in two of these Sendai virus and E. cuniculi antibodies were also found. The pet shop guineapigs were infected with adenovirus, reovirus type 3 and E. cuniculi.

Influenza virus nucleoprotein-specific immunoglobulin G subclass and cytokine responses elicited by DNA vaccination are dependent on the route of vector DNA delivery.

Endpoint immunoglobulin G (IgG) titers and cytotoxic T-lymphocyte (CTL) activities were identical between mice immunized via the intramuscular and epidermal (gene gun) routes with 100 and 1 micrograms, respectively, of an influenza virus nucleoprotein (NP) expression vector. However, examination of the relative levels of two IgG subclasses demonstrated that muscle inoculation resulted in predominantly IgG2a responses, whereas gene gun immunization yielded a preponderance of IgG1 antibodies. Inasmuch as these data suggested that muscle inoculation and gene gun delivery elicited Th1-like and Th2-like responses, respectively, gamma interferon release profiles from antigen-stimulated splenocytes were remarkably similar between these groups. Interleukin-4 (IL-4) production assays, on the other hand, revealed qualitative differences that could be correlated with the divergent IgG subclass data. Waning gamma interferon production in gene gun-immunized animals was countered by a marked increase in IL-4 production following the third immunization, as was the case in control animals immunized with inactivated influenza virus formulated with Freund's adjuvant. In contrast, significant levels of IL-4 production were not observed in the intramuscular DNA inoculation group, despite similar decreases in gamma interferon production with increasing immunizations. These data show that intramuscular inoculation leads to Th1-like responses due to elevated IgG2a levels, production of gamma interferon, CTL activity, and lack of IL-4. However, gene gun responses are more difficult to categorize because of the presence of significant gamma interferon and CTL activity on the one hand and elevated IgG1 antibodies and increasing IL-4 production with successive immunizations on the other. In addition, there was a lack of correlation between IgG isotype ratios and cytokine production in all of the NP DNA-immunized animals, in that IgG subclass ratios remained fixed while cytokine production patterns fluctuated with successive immunizations. These data are consistent with the idea that the types of responses elicited following DNA immunization. are dependent on both the identity of the antigen and the route of DNA administration.

Kinetics, localization and isotype profile of antibody responses to immune stimulating complexes (iscoms) containing human influenza virus envelope glycoproteins.

The immune stimulating complex (iscom) is a particulate adjuvant formulation combining multimeric presentation of antigen with a built-in adjuvant, Quillaja saponin. Iscoms induce strong serum antibody responses that are readily boosted. To further characterize this property of iscoms, the development and maturation of primary and secondary antibody responses to iscoms containing influenza virus antigen were investigated, in serum by ELISA and on single B-cell level by ELISPOT. After a single subcutaneous injection, B cells secreting antigen-specific IgG (IgG-SC) were primarily observed in the draining lymph nodes (LN), showing peak numbers at day 7 which then declined rapidly. Serum IgG levels, as well as IgG-SC in the spleen, persisted for several weeks and, with time, IgG-SC cells also appeared in the bone marrow (BM). These results suggest that the IgG response to iscoms initially is located to the LN but that IgG-SC are redistributed with time and may persist for a long time in other organs, including the spleen and BM. Moreover, a booster dramatically enhanced the frequency of IgG-SC in LN, spleen and BM suggesting that iscoms induce a potent B-cell memory. Comparisons of antibody responses to iscoms with those to influenza virus antigen in Freund's complete adjuvant, TiterMax or aluminium hydroxide suggest that the choice of adjuvant influences both the magnitude, kinetics, localization and isotype profile of antibody responses.

Immunoglobulin G subclass antibodies to rubella virus in chronic liver disease, acute rubella and healthy controls.

Ten patients with chronic liver disease, seven healthy seropositive individuals with a remote history of rubella, and three patients with acute rubella were examined for serum levels of IgG subclasses and subclass antibodies against rubella virus structural proteins. One patient with AICAH had no detectable total or rubella specific IgG3 or IgG4. The liver disease patients were hypergammaglobulinemic and had greatly raised IgG1 levels. Patients with acute rubella lacked antibodies to the rubella virus E2 protein and showed no IgG4 antibody response. The liver disease patients showed a somewhat weaker IgG4 antibody response against the core (C) protein than healthy controls. However, differences are suggested within the subclasses in antibody reactivity against the individual rubella virus antigens. It is concluded that test systems that discriminate reactivities against individual antigens have to be used for characterization of viral antibody subclass profiles.

Determination of the concentration of influenza virus antihaemagglutinin antibody molecules of the IgG class and of the equilibrium constant by use of enzyme immunoassay titres determined for graded epitope concentrations.

A new technique for determining the concentration of influenza virus antihaemagglutinin antibody molecules of the IgG class (A) and of the equilibrium constant K of paratope-epitope interaction is described. The method is based on determining enzyme immunoassay (EIA) titres of antibody with graded epitope concentrations: EIA titres were defined in terms of the antibody dilution yielding a fixed amount of antibody adsorbed per ml (= 6.21 x 10(10)). Adsorption of antibody depends on the concentration of paratopes and epitopes allowed to react and on the equilibrium constant. For the use of a constant concentration of epitopes, the paratope concentration needed to yield the desired degree of antibody adsorption decreases with increasing avidity. Therefore, the EIA titres increase both with increasing avidity and increasing antibody concentration. When graded epitope concentrations are used for determining the EIA titres of a given serum, the titres are influenced in a similar manner by the antibody concentration of the serum and the increase of titres with increasing epitope concentration reflects avidity. The equilibrium constant found is subsequently used to determine the concentration of free antibody at the dilution meeting the definition of EIA titre and the product of EIA titre and the sum of free and bound antibody at this dilution gives the number of antibody molecules present in the test serum. A panel of 118 antisera was tested comparatively for A and K using the new method and by means of the guanidine titre ratio test and equilibrium filtration. The values obtained agreed well with each other. This novel technique offers the advantage that it can be easily adapted for use with other viruses.

Peptide polymerisation facilitates incorporation into ISCOMs and increases antigen-specific IgG2a production.

Synthetic peptides can be tailor-made to include any B or T epitopes desired from a single or multiple antigens or organisms. However, peptides in general are not very immunogenic and have not proven easy to incorporate into immunogenic vaccines. ISCOMs is an adjuvant system that has the capability not only to enhance the humoral immunogenicity of a protein but has also been shown to induce cell-mediated immune responses in animals. Synthetic peptide ISCOM vaccines are few because of the difficulty in incorporation of these peptides into ISCOMs. We have shown in this study that non-immunogenic peptides could be made immunogenic by polymerisation, and these polymers could be incorporated into ISCOMs to give highly immunogenic vaccines. Synthetic 20mer peptides containing known B and T-helper epitopes from the E7 protein of the cervical cancer associated human papillomavirus type 16 (HPV16 E7) have been used here as model immunogens. We have compared the humoral immunity induced by these peptides as polymers or as copolymers with a lipid binding 20mer peptide (LAP 20), with or without incorporation into ISCOMs. Unpolymerised peptide elicited no measurable antibody. When polymerised peptide was administered with CFA, or in phosphate-buffered saline (PBS) without adjuvant, or incorporated into ISCOMs, antibodies recognising both the immunising peptide and HPV16 E7 protein were produced. For equal quantities of administered peptide (5 micrograms), ISCOMs gave higher titres of antibody than CFA or PBS. Polymerised peptides induced high antigen-specific IgG2a:IgG1 ratios, which increased with multiple immunisations. These data indicate that polymerised peptides could be incorporated into ISCOMs to form efficient immunogens which may elicit a Th1 type response.

Similar subclass antibody responses after intranasal immunization with UV-inactivated RSV mixed with cholera toxin or live RSV.

To determine the effect of cholera toxin as a mucosal adjuvant on the class and subclass antibody response to RSV, mice were immunized intranasally with different doses of live RSV or UV-inactivated RSV mixed with cholera toxin. A single 10(6) pfu dose of live RSV and a single 50 micrograms dose of UV-inactivated RSV mixed with cholera toxin produced comparable serum IgG and respiratory secretion IgG and IgA antibody titers. Subclass antibody titers to whole RSV were also comparable between these two immunizing regimens. A predominance of IgG2a subclass to whole RSV was found for both regimens. The quantity of serum total IgG antibody to glycoprotein F or glycoprotein G did not differ between these regimens. The serum IgG subclass antibody response to both glycoprotein F and G was also not significantly different between regimens. Cholera toxin as a mucosal adjuvant can stimulate class and subclass antibody responses to UV-inactivated RSV that are similar in quantity and distribution to those after live RSV infection.