Distinct cellular level of the Ca2+ binding chaperone calreticulin (CRT) is essential for cardiac development and postnatal function. However, CRT is also a potential autoantigen eliciting formation of antibodies (Ab), whose role is not yet clarified. Immunization with CRT leads to cardiac injury, and overexpression of CRT in cardiomyocytes induces dilated cardiomyopathy (DCM) in experimental animals. Hence, we analysed levels of anti-CRT Ab and calreticulin in the sera of patients with idiopatic DCM and hypertrophic cardiomyopathy (HCM). ELISA and immunoblot using human recombinant CRT and Pepscan with synthetic, overlapping decapeptides of CRT were used to detect anti-CRT Ab. Significantly increased levels of anti-CRT Ab of IgA (P<0.001) and IgG (P<0.05) isotypes were found in patients with both DCM (12/34 seropositive for IgA, 7/34 for IgG) and HCM (13/38 seropositive for IgA, 11/38 for IgG) when compared with controls (2/79 for IgA, 1/79 for IgG). Titration analysis in seropositive DCM and HCM patients documented anti-CRT Ab detected at 1/1600 dilution for IgG and 1/800 for IgA (and IgA1) and at least at 1/200 dilution for IgA2, IgG1, IgG2 and IgG3. Pepscan identified several immunogenic CRT epitopes: EVKIDNSQVESGSLED, IDDPTDSKPE, DKAPEHIPDPDA and RKEEEEAEDKEDDAEDKDEDEEDE recognised by IgA and...

The gonococcal transferrin binding proteins (Tbps) are two surface-exposed outer membrane proteins, TbpA and TbpB, which together function to remove and internalized iron from human transferrin. Iron is an essential nutrient to the gonococcus, without which it cannot survive. The Tbps have been established as virulence factors, demonstrating their importance in establishing infection. Both TbpA and TbpB are well conserved among gonococcal isolates, and have been considered potential vaccine targets. Vaccine studies with the closely related species Neisseria meningitidis, have demonstrated these proteins to be protective in murine challenge studies. Though the meningococcal Tbps have demonstrated promise, no similar gonococcal vaccine experiments have been conducted prior to the current studies. Here we demonstrate purification of recombinant TbpA and TbpB. These recombinant proteins were utilized to evaluate the human immune response to these proteins during natural infections, and their immunogenicity in murine vaccine studies. Our results demonstrate a paucity of antibodies elicited to these proteins during natural infections in serum and mucosal secretions from infected individuals. From this study we hypothesized the induction of both serum and genital antibodies to these proteins could serve to protect an individual from infection. To begin testing this hypothesis, we immunized mice both intranasally (IN) and subcutaneously (s.c.) with full-length Tbps in conjunction with the B subunit of cholera toxin (Ctb) as an adjuvant. We also performed another vaccine study using domains from both proteins in genetic fusions with Ctb and E. coli heat labile toxin IIb (LtbIIb). Both studies demonstrated that these antigens were immunogenic, as Tbp-specific antibodies were elicited in the serum and vaginal washes of female Balb/C mice. Intranasal immunization however was the only route with which we were able to elicit vaginal Tbp-specific IgA, and IgG, whereas subcutaneous immunization only elicited vaginal IgG. Furthermore, we found the full-length Tbps and the Ctb/LtbIIb chimeras were able to elicit bactericidal antibodies, which were also effective in killing heterologous gonococcal strains. This body of work comprises the first published study using the gonococcal transferrin binding proteins as vaccine antigens, and highlights their potential as vaccine antigens in the development of an efficacious gonococcal vaccine.

Our understanding of humoral immune response to human papillomavirus (HPV) infection has been mainly derived from studies in women. Very little is known about humoral immune response to HPV in men. There is also a growing interest in understanding the burden of HPV exposure in the subgroups of the male population, including men who have sex with women (MSW), men who have sex with men (MSM) and men who have sex with both men and women (MSMW). This dissertation was undertaken to understand and characterize humoral immune response, measured by detectable serum antibody IgG, to HPV 6, 11, 16 and 18 infection, to estimates seroprevalence of HPV 6, 11, 16 and 18, to determine the associations of sociodemographic and sexual behavioral factors with seroprevalence of individual HPV types, and to evaluate the role of serum antibodies in the subsequent acquisition of infection with the same HPV type, genetically related and un-related HPV types. Three studies that compose of this dissertation were conducted within the framework of two longitudinal studies of HPV infection in men: a single-site natural history study of male residents of Tucson, Arizona (the 1st study: N=285); and a multinational natural history study of healthy men residing in São Paulo, Brazil, Cuernavaca, Mexico, and Tampa, Florida (the 2nd study: N=1477; the 3rd study: N=2187). Men were recruited using similar eligibility criteria in both natural history studies and followed every 6 months for a maximum of 18 months in the single-site study and 48 months in the multi-national study. HPV DNA status was assessed using the PGMY09/11 L1 consensus primer system and the Linear Array HPV Genotyping Protocol. Testing of serum antibodies to HPV 6, 11, 16 and 18 was performed with virus-like particle-based ELISA assays. Data from our studies indicate that exposure to HPV 6, 11, 16 and 18, the four HPV types targeted in the currently license HPV vaccines, is common. Of 285 male residents of Tucson, Arizona, 28.8% of them were seropositive to HPV 16 and/or 18 at study entry. Similarly, approximately one third of 1477 participants of the multi-national male HPV natural history study were seropositive to at least one vaccine HPV type, with the percentage of 21.8% in U.S. site, 33.4% in Mexico site, and 49.1% in Brazil site. It is also noted that seroprevalence of individual vaccine HPV types is greatly elevated among men of different sexual practices. Seroprevalence of HPV 6, 11, 16 and/or 18 was twice as high among MSM and MSMW compared to MSW. Likewise, seroprevalence of individual HPV types was two fold or higher among MSW and MSMW. Our findings suggest that the predominant predictors of seropositivity to HPV 6, 11, 16 and 18 are age and same-sex sexual behaviors. Seroprevalence increased with age among young-to-middle-aged men with significant upward age trends observed for HPV 11, 16 and 18. MSM, compared to MSW, more likely to be seropositive to HPV 16 or 18. Similarly, men who practiced same-sex anal sex, compared to those who did not, were significantly more likely to be seropositive to HPV 6, 11, 16 and 18, respectively. Among 276 men free of HPV 16 at enrollment in Tucson, We did not detect statistically significant associations between the baseline serum antibodies to HPV 16 and/or 18 and subsequent risk of infection with homogeneous HPV types or related-HPV types. Of 2187 men residing in three countries who tested HPV 16 negative at enrollment, the risk of subsequent HPV 16 infection was not associated with enrollment HPV 16 serum antibodies status. Our data provide important estimates of population exposure to vaccine HPV types for future studies modeling potential vaccine impact and vaccine cost effectiveness in men. Our findings also support strategic vaccination of males as an effective preventive measure for HPV-related diseases and cancers in men and their sex partners, men and women alike.