No abstract

Development of vaccines capable of preventing the transmission or limiting the severity of sexually transmitted viruses, such as HSV and HIV, will likely be dependent on the induction of potent long-lasting mucosal immune responses in the genital tract. Recently, synthetic oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs were shown to serve as potent adjuvants for the induction of mucosal immune responses. Here, we show that intranasal immunization with CpG ODN, plus recombinant glycoprotein B (rgB) of HSV-1, results in significantly elevated levels of specific anti-gB IgA Abs in vaginal washes that remained high throughout the estrous cycle. Additionally, dramatically elevated numbers of specific IgA Ab-secreting cells were present and persisted in the genital tract in response to intravaginal (IVAG) HSV-2 challenge. HSV-2-specific CTL were observed at moderate levels in the spleens of CpG or non-CpG ODN-immunized mice. In contrast, strong CTL responses were observed locally in the genital tissues of both groups following IVAG HSV-2 challenge. Interestingly, mice immunized intranasally with rgB plus CpG ODN, but not non-CpG ODN, were significantly protected following IVAG HSV-2 challenge. Measurement of virus in protected CpG-immunized mice revealed a log lower level of replication within the first few days after infection. In conclusion, these results indicate that intranasal immunization with CpG ODN plus protein mediates immunity in the female genital tract capable of protecting against a sexually transmitted pathogen.

Vaccination of two chimpanzees against hepatitis B virus (HBV) by intramuscular injection of plasmid DNA encoding the major and middle HBV envelope proteins induced group-, subtype-and preS2-specific antibodies. These were initially of IgM isotype, and then they were of IgG (predominantly IgGl) isotype. The chimpanzee injected with 2 mg of DNA attained >100 milli-international units/ml of anti-HBs antibody after one injection and 14,000 milliinternational units/ml after four injections. A smaller dose (400 ,ug) induced lower and transient titers, but a strong anamnestic response occurred 1 year later. Comparison with responses in 23 chimpanzees receiving various antigen-based HBV vaccines suggests that the DNA approach is promising for prophylactic immunization against HBV.Hepatitis B virus (HBV) remains an important worldwide health problem, with an estimated 250 million chronic carriers who face increased risk of developing cirrhosis and hepatocellular carcinoma (1). The prospects for control of infection and disease depend on the availability of safe, effective, and affordable vaccines.Although both humoral and cell-mediated immunity may result from natural HBV infection, antibodies alone are sufficient to confer protection, and the exact role of cytotoxic T lymphocytes is not known. After natural infection, antibodies are detected against the surface antigen of the HBV viral envelope (HBsAg; anti-HBs) and the viral core protein (HBcAg; anti-HBc/anti-HBe). There is a very clear role for anti-HBs in conferring protective immunity, and all licensed vaccines used in humans to date have been designed to elicit this. The common clinical standard for anti-HBs antibody levels is milli-international units (mIU)/ml, and in humans, a level of 10 mIU/ml is considered sufficient to confer protection (2). The role for anti-HBc in conferring protective immunity is less clear. Although HBcAg is highly immunogenic and immunization with HBcAg alone has been shown to protect chimpanzees against challenge with live HBV (3, 4), high titers of maternal anti-HBc fail to protect infants of chronically infected mothers from infection (4).The structural gene for the HBV envelope protein is a single long open reading frame containing three inframe ATG start codons (dividing the gene into three domains designated preS1, preS2, and S from 5' to 3') and a single stop codon. The different sized polypeptides produced are known as small or major (S), middle (M = preS2 + S), and large (L = preSl + preS2 + S). The envelope of the infectious 42 nm HBV (Dane) particle contains all forms, but with a predominance of S. The serum of infected individuals also contains large numbers of smaller (22 nm) empty subviral particles composed solely or predominantly of S (5). B-and T-cell epitopes are found on both S and preS domains. The S domain encodes the primary protective B-cell epitope (group-specific determinant a) and two other determinants, (d or y and w or r), resulting in four subtypes: adw, adr, ayw, and ayr (1).The first HBV vaccines ...

Oligodeoxynucleotides (ODN) with unmethylated CpG dinucleotides mimic the immune stimulatory activity of bacterial DNA in vertebrates and are recognized by Toll-like receptor 9 (TLR9). It is also possible to detect immune activation with certain phosphorothioate sequences that lack CpG motifs. These ODN are less potent than CpG ODN and the mechanism by which they stimulate mammalian leucocytes is not understood. We here provide several lines of evidence demonstrating that the effects induced by non-CpG ODN are mediated by TLR9. First, non-CpG ODN could not stimulate cytokine secretion from the splenocytes of TLR9-deficient (TLR9(-/-)) mice. Second, immunization of TLR9(+/+) but not TLR9(-/-) mice with non-CpG ODN enhanced antigen-specific antibody responses, although these were T helper type 2 (Th2)-biased. Third, reactivity to non-CpG ODN could be reconstituted by transfection of human TLR9 into non-responsive cells. In addition, we define a new efficient immune stimulatory motif aside from the CpG dinucleotide that consists of a 5'-TC dinucleotide in a thymidine-rich background. Non-CpG ODN containing this motif induced activation of human B cells, but lacked stimulation of Th1-like cytokines and chemokines. Our study indicates that TLR9 can mediate either efficient Th1- or Th2-dominated effects depending on whether it is stimulated by CpG or certain non-CpG ODN.

No abstract

Background: In spite of the large number of studies that have evaluated DNA-based immunization, few have directly compared the iimmune responses generated by different routes of immunization, particularly in nonhuman primates. Here we examine the ability of a hepatitis B surface antigen (HBsAg)-encoding plasmid to induce immune responses in mice and non-human primates (rhesus monkeys: Macaca mulatta) after delivery by a number of routes. Materials and Methods: Eight different injected [intraperitoneal (IP), intradermal (ID), intravenous (IV), intramuscular (IM), intraperineal (IPER), subcutaneous (SC), sublingual (SL), vaginal wall (VW)] and six noninjected [intranasal inhalation (INH), intranasal instilla-tion (INS), intrarectal (LR), intravaginal (IVAG), ocular (Oc), oral feeding (oral)] routes and the gene gun (GG) were used to deliver HBsAg-expressing plasmid DNA to BALB/c mice. Sera were assessed for HBsAg-specific antibodies (anti-HBs, IgG, IgGI, IgG2a) and cytotoxic T lymphocyte (CTL) activity measured. Three of the most commonly used routes (IM, ID, GG) were compared in rhesus monkeys, also using HBsAg-expressing vectors. Monkeys were immunized with short (0-, 4-and 8-week) or long (0-, 12-and 24-week) intervals between boosts, and in the case of GG, also with different doses, and their sera were assessed for anti-HBs. Results: In one study, anti-HBs were detected in plasma of mice treated by five of eight of the in-DNA IM or ID at 0, 4, and 8 weeks gave equivalent anti-HB titers and 0.4 ,jg at the same times by GG induced lower titers. In the second experiment, 1 mg DNA IM or ID, or 3.2 jig by GG, at 0, 12, and 24 weeks, gave anti-HB values in the hierarchy of GG > IM > ID. Furthermore, high titers were retained after a single immurnization in mice but fell off over time in the monkeys, even after boost. Conclusions: Route of administration of plasmid DNA vaccines influences the strength and nature of immune responses in mice and non-human primates. However, the results in mice were not always predictive of those in monkeys and this is likely true for humans as well. Optimal dose and immunization schedule will most likely vary between species. It is not clear whether results in non-human primates will be predictive of results in humans, thus additional studies are required.

Anti-nicotine vaccines may aid smoking cessation via the induction of anti-nicotine antibodies (Ab) which reduce nicotine entering the brain, and hence the associated reward. Ab function depends on both the quantity (titer) and the quality (affinity) of the Ab. Anti-nicotine vaccines tested previously in clinical studies had poor efficacy despite high Ab titer, and this may be due to inadequate function if Ab of low affinity were induced. In this study, we designed and synthesized a series of novel nicotine-like haptens which were all linked to diphtheria toxoid (DT) as carrier, but which differed in the site of attachment of linker to nicotine, the nature of linker used, and the handle used to attach the hapten to DT. The resulting hapten conjugates were evaluated in a mouse model, using CpG (a TLR9 agonist) and aluminum hydroxide (Al(OH)3) as adjuvants, whereby Ab titers, affinity and function were evaluated using a radiolabeled nicotine challenge model. A series of additional linkers varying in length, rigidity and polarity were used with a single hapten to generate additional DT-conjugates, which were also tested in mice. Conjugates made with different haptens resulted in various titers of anti-nicotine Ab. Several haptens gave similarly high Ab titers, but among these, Ab affinity and hence function varied considerably. Linker also influenced Ab titer, affinity and function. These results demonstrate that immune responses induced in mice by nicotine-conjugate antigens are greatly influenced by hapten design including site of attachment of linker to nicotine, the nature of linker used, and the handle used to attach the hapten to DT. While both Ab titer and affinity contributed to function, affinity was more sensitive to antigen differences.

No abstract